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Mechanism of ionophore A23187 induction of plasma protein leakage and of its inhibition by indomethacin
European Journal of Pharmacology, 81 (1982) 35-42 Elsevier Biomedical Press
35
MECHANISM OF IONOPHORE A23187 INDUCTION OF PLASMA PROTEIN LEAKAGE AND OF ITS INHIBITION BY INDOMETHACIN G. THOMAS
Laboratbrio de Tecnologia Farmac~utica, Universidade Federal da Paraiba, 58.000 Jo~o Pessoa, Paraiba, Brazil Received 8 September 1981, revised MS received 19 January 1982, accepted 1 March 1982
G. THOMAS, Mechanism of ionophore A23187 induction of plasma protein leakage and of its inhibition by indomethacin, European J. Pharmacol. 81 (1982) 35-42. Calcium ionophore A23187 produced a dose-dependent increase in plasma protein leakage on intradermal injection in rats. Studies with mepyramine and cyproheptadine indicated that histamine and 5-hydroxytryptamine (5-HT) partially contribute to the ionophore action and experiments with compound 48/80 supported these findings. Depletion of plasma kininogen levels with cellulose sulphate did not significantly inhibit the ionophore effect in rats suggesting that kinins are unimportant in the ionophore action. Orally administered indomethacin inhibited the ionophore response in a dose-dependent manner. The inhibition was not reversed by intradermally injected prostaglandin E 2 (PGE2) in doses up to 50 ng/site, suggesting that PGE 2 also may not be an important mediator. It is proposed that the ionophore produces plasma protein leakage by an indirect (through histamine and 5-HT release) and a direct action on vascular endothelial cells and that indomethacin antagonises both actions by inhibiting calcium transport processes. Ionophore A23187
Protein leakage
Mediators
Indomethacin
1. Introduction
Ionophore A23187 produces diverse pharmacological actions by its ability to transport divalent cations, preferentially calcium ions, across cell membranes (Pressman, 1973). Some of these recently reported actions include secretion of endocrine hormones (Pressman, 1976; Heisler, 1976), contraction of smooth muscle (Watson, 1978; Ishida and Shibata, 1980), stimulation of release of neurotransmitters (Casamenti et al., 1978; Ito et al., 1978), and promotion of axoplasmic transport of dopamine fl-hydroxylase (Esquerro et al., 1980). The ionophore was also shown to cause the release of histamine from rat mast cells (Foreman et al., 1973; Johansen, 1979), of SRS-A from human leucocytes (Conroy et al., 1976) and human and guinea-pig lungs (Piper and Seale, 1978), to stimulate the synthesis of thromboxane B2 and prostaglandins in rat polymorphonuclear leucocytes (PMNs) (Knapp et al., 1977) and the production of chemokinetic factors from human PMNs (Bray et al., 1980). 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
The objective of the present work was to determine whether ionophore A23187 has the ability to produce vascular permeability changes on intradermal injections into rats and if so, to study the mediators involved in this effect in comparison to the action of dextran, an agent known to produce its effect by releasing mainly 5-HT and histamine (Parratt and West, 1958). In studies conducted in vitro, the dextran response was also found to depend on calcium ions (Foreman and Mongar, 1972). Additional experiments were performed to observe whether indomethacin has antagonistic effects on the ionophore A23187 response, since other calcium-dependent effects of the ionophore, such as release of mast cell histamine and contraction of guinea-pig ileum, are inhibited by indomethacin (Lewis and Whittle, 1977; Thomas, 1980). Further, it has also been reported that indomethacin interferes with the participation of calcium ions in many other biological processes (Northover, 1977; Continenza et al., 1980).
36 2. Materials and methods
2.1. Vascular permeability studies with ionophore A23187 Male rats weighing 150-180g had their backs shaved 48 h before being used in experiments. Initially, pentobarbitone (40 m g / k g i.p.) was used as the anaesthetic agent. However, the ionophore was found to produce greater responses when ether was used and all subsequent experiments were therefore performed under ether anaesthesia. After anaesthetising the animals, Evans blue (20 mg/kg) was injected i.v. followed by i.d. injections of concentrations of the ionophore into the shaved areas, in dose volumes of 0.1 ml. A 2 m g / m l stock solution of the ionophore was prepared in ethanol, kept at - 18°C and diluted with saline as required. As each dose of the ionophore injected contained ethanol (the amount depending on the concentration of the ionophore), appropriate concentrations of ethanol in saline were also injected into different sites. Since maximal effects were produced between 30-45 rain after the ionophore applications, the rats were killed 45 min after the i.d. injections and the intensity of plasma protein leakage measured spectrophotometrically by estimating the amount of dye in each weal according to the method of Harada et al. (1971). The effects of the ionophore alone were obtained by subtracting the amount of dye exuded when diluted solutions of ethanol were injected from the appropriate responses to the ionophore. 2.2. Studies on mediators of ionophore A23187 response 2.2.1. Experiments with antagonistic drugs For the experiments with mepyramine, the dose of antagonist that inhibited (70-80%) the histamine response in the skin of rats was first established by injecting mepyramine i.p. 30 rain before the i.d. administration of a concentration of histamine that produced a submaximal response. This dose of mepyramine was administered i.p. into a group of 6 rats, followed 30 min later by i.d. injections of histamine, 5-HT, dextran and the ionophore. The doses of the agonists were such that they produced approximately similar skin
responses. Another group of 6 rats receiving saline i.p. served as control. Both groups received appropriate i.d. injections of ethanol and saline as well. The amount of dye which leaked at each site of injection was estimated as described earlier. Percent inhibition was calculated by comparing the responses (after subtracting the corresponding values for saline and ethanol injections) using the formula: [ ( R c - R t ) / R c ] X 100 where Rc and Rt are mean responses to agents in the saline- and mepyramine-treated groups respectively. When cyproheptadine was used as the antagonist, a concentration of cyproheptadine which inhibited the 5-HT response by more than 70% was administered i.p. 30 min before i.d. injections of histamine, 5-HT, dextran and the ionophore. Percent inhibition of responses for each agent was calculated as before. The effects of giving mepyramine and cyproheptadine simultaneously on the ionophore response were studied by giving both the antagonists in the doses used earlier and injecting the ionophore 30 min later. 2.2.2. Depletion studies Using compound 48/80, rats were depleted of their stores of histamine and 5-HT according to the schedule described by Di Rosa et al. (1971). Other rats receiving saline injections served as control. Both groups received i.d. injections of submaximal doses of dextran and the ionophore A23187 and the amount of dye exuded was estimated and the percent inhibition calculated as described before. Rats were depleted of plasma kininogen by repeated i.v. injections of cellulose sulphate as described by Di Rosa and Sorrentino (1970). This method lowers plasma kininogen levels by about 50%. The rats were used three hours after the last dose of cellulose sulphate for i.d. injections of dextran and ionophore. 2.3. Studies with indomethacin Giving indomethacin 12-24h before the ionophore injections was found to produce the most
37
marked inhibition. Therefore, doses of indomethacin were given orally to rats, 18 and 1 h before i.d. application of a selected submaximal dose of the ionophore. Three injections of ionophore and one injection of the vehicle were made in each animal. The effect of indomethacin on the protein exudation induced by the ionophore was determined by comparing the mean ionophore response in the control- and drug-treated groups. In a few experiments, doses of 10 or 50 ng/site of PGE 2 were also injected i.d. together with the ionophore to find whether the action of indomethacin (2 × 3.4 mg/kg) could be modified by externally applied PGE 2. Similar concentrations of PGE 2 alone were also injected to determine the effect of indomethacin on PGE 2 responses.
ing appropriate concentrations of ethanol in saline are also shown. Ionophore A23187 caused a concentration-dependent increase in plasma protein leakage, the largest dose of 21 #g/site producing an intense reaction spreading over a large area of the skin surrounding the site of injection. However, part of this response was due to the vehicle, a 12.5% solution of ethanol in saline which also produced marked dye leakage. Table 1 also demonstrates that the ionophore produced greater responses when ether was used as the anaesthetic agent in the animals. However, the results were significantly (P < 0.05) different only for 9 #g/site of the ionophore. Since injection of 9#g/site caused an easily measurable consistent response which was minimally affected by the vehicle (4.5% ethanol), this dose was selected for all subsequent experiments with the ionophore.
3. Results
3.1. Plasma protein leakage induced by the ionophore A23187 Table 1 shows the effects of i.d. injections of calcium ionophore A23187 on plasma protein leakage in rats under ether or under pentobarbitone anaesthesia. The results obtained after inject-
3.2. Mediators of ionophore A23187 response in the skin of rats 3.2.1. Experiments with antagonistic drugs The inhibitory effects of mepyramine and cyproheptadine on the ionophore response are shown in table2. A dose of 2.5 mg/kg i.p. of mepyramine significantly inhibited the responses
TABLE 1 Increase in plasma protein-bound dye leakage induced by i.d. injections of ionophore A23187 in ethanol and of 0.1 ml/site of appropriately diluted solutions of ethanol alone into groups of rats anaesthetized with either pentobarbitone or ether. Dye leakage (pg) was measured 45 min after the injections. Dose of ionophore and/~g of dye exuded
Concentration of ethanol and #g of dye exuded
Dose ~g
/~g of dye (A)"
% ethanol
Pentobarbitone
i.0 3.0 9.0 27.0
7.4±1.5" 10.1~1.1"* 19.0±1.3"** 30.0±4.1"*
0.5 1.5 4.5 12.5
2.6±0.1 2.0±0.1 3.5±0.2 7.6±1.1
4.8 8.1 15.5 22.4
Ether
1.0 3.0 9.0 27.0
9.9±2.0* 12.6±1.2"** 27.0±2.2*** 43.8±6.0**
0.5 1.5 4.5 12.5
2.5±0.1 3.0±0.3 3.5±0.3 11.0±2.5
7.4 9.6 23.5 32.8
Anaesthetic
/~g of dye (E)a
Dye exuded (#g) with ionophore alone (A--E)
Student's t-test: * P<0.025, ** P
38 TABLE 2 Antagonistic actions of mepyramine (2.5 m g / k g ) and cyproheptadine (2.5 m g / k g ) on plasma protein leakage induced by i.d. injections of histamine (10/~g), 5-HT (250 ng), dextran (9/~g) and ionophore A23187 (9 ~g) in groups of 6 rats. The mean amount of dye (/Lg/site) exuded with each agonist was measured 45 min after the i.d. injections. Agonist
Histamine 5-HT Dextran Ionophore A23187
/~g (mean ±S.E.M.) of dye e x u d e d / s i t e after pretreatment with
% inhibition of dye leakage with
Saline
Mepyramine
Mepyramine
Cyproheptadine
28.8 --+3.7 23.0--+3.5 30.0±3.6
6.8 + 1.1 *** 17.5± 1.5 NS 19.0+2.0 *
75.7 24.0 36.7
76.7 85.7 86.3
-
31.8--+4.0
25.2±4.0 ys
20.8
30.8
47.5
Cyproheptadine
Mepyramine + Cyproheptadine
6.7 ± 0.9 *** 3 . 3 ± 0 . 4 *** 4.1 +0.5 ***
-
22.0±2.2 *
16.7 + 1.2 **
Mepyramine + cyproheptadine
-
Student's t-test: * P < 0 . 0 5 , ** P<0.01, *** P<0.001. ys Not significant at the 95% probability level.
to histamine (P < 0.001) and dextran (P < 0.05). Mepyramine inhibited the responses to histamine, dextran, 5-HT and the ionophore by 75.7, 36.7, 24.0 and 20.8% respectively. Cyproheptadine in a dose of 2.5 mg/kg i.p. significantly antagonised the effects of histamine (P<0.005), 5-HT ( P < 0.001), dextran (P < 0.001) and the ionophore (P < 0.05). However, the degree of antagonism of the ionophore response (30.8%) was much less than that of the 5-HT, histamine and dextran responses (85.7, 76.7 and 86.3%, respectively). The combina-
tion of mepyramine and cyproheptadine also failed to inhibit the ionophore action to any great extent as the inhibition was only 47.5% suggesting that mechanisms other than histamine and 5-HT release contribute to the effect of the ionophore A23187 in the skin of rats.
3. 2.2. results of depletion studies The dextran response was significantly ( P < 0.001) reduced (79.1%) in compound 48/80pre-treated animals (table 3). Similar pre-treatment
TABLE 3 Effect of pretreatment of rats with compound 4 8 / 8 0 or cellulose sulphate on vascular permeability changes induced by intradermal injections of dextran (9 #g) and ionophore A23187 (9/lg) in groups in 6 rats. The mean amount (/~g/site) of dye exuded with each agent was determined 45 min after the i.d. injections. Test Agent
# g (mean - S . E . M . ) of dye e x u d e d / s i t e after pretreatment with Saline
Compound 48/80
Test I Dextran lonophore
25.3 ± 1.9 20.3±3.6
5.3 -4- 1.0 *** 12.2 + 1.1 *
Test H Dextran Ionophore
26.2 ± 1.6 22.0 ± 2.0
-
% inhibition of dye leakage with Cellulose sulphate
23.0 ± 3.0 NS 18.5 ± 2.1 NS
Student's t-test: * P<0 .0 5, *** P<0.001. NS Not significant at the 95% probability level.
Compound 48,/80
Cellulose sulphate
79.1 39.9
-
_ _
12.3 15.9
39 TABLE 4 Effect of graded doses of indomethacin on ionophore A23187-induced dye leakage in the skin of groups of 6 rats. Indomethacin was administered orally twice: 18 and 1 h before the injections of the ionophore. Dye leakage was estimated 45 min after the i.d. injections. Drug
Dose mg/kg p.o.
/,tg of dye exuded (mean ± S.E.M.)
Saline
10ml
20.5±2.6
1.25 2.5 5.0 10.0
16.5-+1.8 Ns 11.4±1.1" 8.3±1.9"* 3.3±1.0"**
% inhibition
EDs0 mg/kg
19.5 ~.3 59.5 83.9
3.4
Indomethacin
Student's t-test: * P < 0 . 0 5 , ** P<0.01, *** P<0.001. NS Not significant at the 95% probability level.
3.3. Inhibitory studies with indomethacin
with compound 48/80 also inhibited the ionophore effect in a significant (P < 0.05) manner, but the inhibition (39.1%) was much less than that obtained with the dextran skin reaction. Table 3 also demonstrates that previous depletion of plasma kinins by cellulose sulphate did not significantly reduce either the dextran- or the ionophore-induced protein leakage in rats. The dextran response was inhibited by 12.3% and the ionophore response by 15.9% when compared with saline-treated control animals.
Various doses of indomethacin, each given orally twice, i.e. 18 and 1 h before the i.d. injections of the ionophore, produced dose-related inhibition of the ionophore-induced dye leakage in rats (table 4). The inhibition was statistically significant for the 2.5 (P < 0.05), 5.0 (P < 0.01) and 10 mg/kg (P < 0.001) doses and the approximate EDs0 value calculated from the dose-response curve was 3.4 mg/kg.
TABLE 5 Experiment I demonstrates the inability of PGE 2 (10 n g / s i t e i.d.) to potentiate the increased dye leakage produced by ionophore A23187 (9 # g/site) and also the failure of this dose of PGE 2 to reverse the inhibitory activity of indomethacin (2 × 3.4 m g / k g p.o.) on the ionophore response. Similar results obtained with 50 n g / s i t e of PGE 2 are given in experiment II. Dye exudation was measured 45 min after i.d. injection in groups of 6 rats. Agent injected i.d. and dose
Experiment I P G E 2 10 ng Ionophore 9/~g Ionophore 9/~ g + PGE 2 10 ng Experiment 11 P G E 2 50 ng lonophore 9/~g Ionophore 9 / ~ g + P G E 2 50 ng
/~g (mean ±S.E.M.) of dye exuded after treatment with
% inhibition with Indometbacin
Saline
Indomethacin
2.6--+0.6 22.9±2.1 23.1 ± 2.4
2.1 ± 0 . 4 NS 12.0-+0.8 ** 11.6 ± 0.5 **
19.2 47.6 49.8
5.0± 1.6 21.4±2.0 25.0±2.6
4.1 ± 1.2 NS 12.6± 1.2 * 16.0± 1.0 *
18.0 40.2 36.0
Student's t-test: * P<0.05, ** P<0.01. Ns Not significant at the 95% probability level.
40 The effects of externally applied PGE 2 on the ionophore response are shown in table 5. On i.d. injection, PGE 2 in doses of either 10 or 50 ng/site, produced only minimal dye leakage which was not significantly ( P < 0 . 1 ) affected by indomethacin (2 × 3.4 mg/kg). In the above doses PGE 2 also failed to potentiate the ionophore response when injected together with this agent. The table also shows that PGE 2 did not modify the inhibitory activity of indomethacin on the ionophore-induced dye leakage. Indomethacin produced inhibition of 47.6 and 49.8% in the absence of PGE 2 or with PGE2, respectively. At the larger indomethacin dose (50 ng/site), inhibition was 40.2 and 36.0% in the absence or presence of PGE 2, respectively. In all these cases, inhibition was statistically significant (P < 0.5 or below).
4. Discussion
Intradermal injections of ionophore A23187 produced dose-related increases in plasma protein leakage in the skin of rats. Since doses of 9/xg/site of both dextran and ionophore A23187 induced responses of approximately similar intensity it is possible that the ionophore was equipotent with dextran in producing vascular permeability changes. This can only be confirmed by comparing the dose-response curves of these two agents. For investigating the mechanism of action of A23187, dextran was selected as the reference agent as the mediation of dextran reaction is well known (Parrett and West, 1958) and further, a number of studies comparing dextran and compound A23187 have been reported (Garland and Mongar, 1976; Foreman et al., 1977). Experiments in separate groups of rats with the specific antagonists mepyramine and cyproheptadine, indicated that when compared to dextran, neither histamine nor 5-HT has a major role in the mediation of the ionophore response in the skin of rats. These resuits are further supported by the fact that less than 50% inhibition of the ionophore response was obtained when this agent was injected into rats given both mepyramine and cyproheptadine previously. The doses of antagonists were such that these agents inhibited the responses to specific
agonists by more than 70%. In addition, the 2.5 m g / k g dose of cyproheptadine alone was sufficient to produce almost complete inhibition of the dextran response. Pre-treatment of rats with compound 48/80 to deplete the stores of histamine and 5-HT in the body suppressed the ionophore response far less than the dextran response, providing further evidence for the relatively minor roles of histamine and 5-HT in the ionophore response. Treatment of rats with cellulose sulphate, a kininogen-depleting agent, failed to inhibit significantly either the dextran- or A23187-induced plasma protein exudation. These results indicate that the action of kinins is not important in this respect even though in other experimental models, kinins have been implicated in the mediation of the dextran response (Garcia Leme et al., 1967; Di Rosa and Sorrentino, 1970). In the present tests PGE 2 did not potentiate the ionophore action even though PGEs have shown such effects in several models (Moncada et al., 1973; Williams and Morley, 1973; Lewis et al., 1975). However, PGE l was also reported to have no potentiating effect on histamine, 5-HT, dextran and compound 48,/80 responses (Jori et al., 1961; Lewis et al., 1975). Lewis et al. (1975) attempted to explain the potentiating effect of PGE 1 and PGE 2 in carrageenan and kaolin-induced rat paw oedema by their ability to act synergistically with kinins released during these responses. Thomas and West (1973) also found that PGE 1 potentiated the bradykinin response while having no effect on the histamine, 5-HT and dextran responses in rat skin. Thus the failure of PGE 2 to modify the ionophore response may be explained partially by the minor role of kinins in this reaction. It has been proposed (Williams and Peck, 1977; Williams, 1979) that an agent produces maximum plasma protein exudation if it has a vasodilatory action in addition to its vascular permeability-increasing property. For example, Wedmore and Williams (1980) demonstrated that three leucotactic agents with minimal intrinsic vasodilatory action produced significant plasma exudation only when PGE z was injected along with these agents. Ionophore A23187 may possess potent vasodilatory properties, in which case the addition of
41
another vasodilator may have no further effect; such an explanation could perhaps also account for the lack of effect of PGE 2 in these experiments. Prostaglandin responses are highly variable in the rat (Freeman and West, 1972) and the intensity of the reaction produced by PGEs even in the maximal doses of 2.5 #g/site is far less than that of the response to compound A23187 and mediators of inflammation (Thomas, 1975). In the present studies, the indomethacin-induced inhibition of the ionophore response was also not reversed by PGE 2 in doses upto 50 ng/site. All these results suggest that PGs may not contribute to the ionophore response in the skin of rats. Further experiments are required to determine the true role of PGs, as calcium ionophores have been shown (Knapp et al., 1977) to stimulate the synthesis of PGs. It is probable that ionophore A23187 causes increased vascular permeability to plasma proteins by a direct action on the vascular endothelial cell as well. Thus, by raising the concentration of intracellular calcium, the ionophore may act on the contractile elements of the cells to produce interendothelial gaps through which plasma proteins may leak. It is proposed that, unlike dextran which acts indirectly by releasing chemical mediators, ionophore A23187 has an additional direct effect in causing plasma protein leakage. Indomethacin produced a dose-dependent inhibition of plasma protein exudation induced by the ionophore. In general non-steroid anti-inflammatory drugs antagonise the vascular permeability changes induced by mediators of inflammation in high doses only, for example, even a dose of 10 mg/kg i.v. of indomethacin failed to inhibited bradykinin-induced dye leakage in rats (Spector and Willoughby, 1968). However, the EDs0 value of 2 × 3.4 mg/kg obtained for indomethacin in the present experiments is close to the EDs0 values of 1.4-2.0 and 2.2-6.5 mg/kg p.o. found in guinea-pig u.v. erythema and rat carrageenan oedema respectively (Scherrer, 1974). Since both indirect and direct actions of the ionophore A23187 depend on transport of extracellular calcium into target cells (Pressman, 1973; Garland and Mongar, 1976; Johansen, 1978; Desmedt and
Hainaut, 1979), the present results support the findings (Famey and Whitehouse, 1976; Northover, 1977; Lewis and Whittle, 1977; Thomas, 1980; Continenza et al., 1980) that indomethacin and other non-steroid anti-inflammatory drugs have effects on calcium transport process in biological membranes. However, the importance of this action in relation to the anti-inflammatory effect of such compounds has yet to be assessed.
Acknowledgment I wish to thank Prof. Delby F. de Medeiros, Director of Laboratrrio de Tecnologia Farmac~utica for his encouragement, Eli Lilly & Co for the gift of ionophore A23187 and Mr Jos~ C. Duarte and Mrs Clebia G. de Oliveira for excellent technical assistance.
References Bray, M.A., A.W. Ford-Hutchinson and M.J.H. Smith, 1980, Generation of chemotactic activity from rat polymorphonuclear leucocytes treated with calcium ionophore A23187, Br. J. Pharmacol. 70, 84 P. Casamenti, F., P. Mantovani and G. Pepeu, 1978, Stimulation of acetylcholine output from brain slices caused by the ionophore Br X-537A and A23187, Br. J. Pharmacol. 63, 259. Conroy, M.C., R.P. Orange and L.M. Lichtenstein, 1976, Release of slow-reacting substance of anaphylaxis (SRS-A) from human leucocytes by the calcium ionophore A23187, J. Immunol. 116, 1677. Continenza, M.A., P. Conti and G.E. Gigante, 1980, Calcium content of experimental granuloma as determined by X-ray fluorescence. Influence of non-steroid anti-inflammatory drugs, Experientia 36, 230. Desmedt, J.E. and K. Hainaut, 1979, Dantrolene and A23187 ionophore: Specific action on calcium channels revealed by the acquorin method, Biochem. Pharmacol. 28, 957. Di Rosa, M., J.P. Giroud and D.A. Willoughby, 1971, Studies of the mediators of the acute inflammatory response induced in rats in different sites by carrageenan and turpentine, J. Pathol. 104, 15. Di Rosa, M. and L. Sorrentino, 1970, Some pharmacodynamic properties of carrageenan in the rat, Br. J. Pharmacol. 38, 214. Esquerro, E., A.G. Garcia and P. Sanchez-Garcia, 1980, The effects of the calcium ionophore A23187 on the axoplasmic transport of dopamine fl-hydroxylase, Br. J. Pharmacol. 70, 375. Famey, J.P. and M.W. Whitehouse, 1976, Effects of nonsteroidal anti-inflammatory drugs on the uptake of various
42 cations by lymphoid cells, Arch. Int. Physiol. Biochem. 84, 719. Foreman, J.C., M.B. Hallet and J.L. Mongar, 1977, The relationship between histamine secretion and 45 calcium uptake by cells, J. Physiol. 271, 193. Foreman, J.C. and J.L. Mongar, 1972, The effect of calcium on dextran induced histamine release from isolated mast cells, Br. J. Pharmacol. 46, 767. Foreman, J.C., J.L. Mongar and B.D. Gomperts, 1973, Calcium ionophores and movement of calcium ions following physiological stimulus to a secretory process, Nature (London) 245, 249. Freeman, P.C. and G.B. West, 1972, Skin reactions to prostaglandins, J. Pharm. Pharmacol. 24, 407. Garcia Leme, J., E.S. Schapoval and M. Rocha e Silva, 1967, Factors influencing the development of local swelling induced in the rat's paw by macromolecular compounds and heating. In Int. Symp. on vasoactive polypeptides: Bradykinin and Related Kinins, eds. M. Rocha e Silva and H.A. Rothschild (Edart, Sao Paulo) p. 213. Garland, L.G. and J.L. Mongar, 1976, Differential histamine release by dextran and the ionophore A23187: the actions of inhibitors, Int. Arch. Allergy Appl. Immunol. 50, 27. Harada, M., M. Takeuchi, T. Fukao and K. Katagiri, 1971, A simple method for the quantitative extraction of dye extravasated into skin, J. Pharm. Pharmacol. 23, 218. Heisler, S., 1976, Calcium and cyclic nucleotide involvement in exocrine pancreatic enzyme secretion studied with the ionophore A23187, Life Sci. 19, 233. Ishida, Y. and S. Shibata, 1980, Characteristic contractile response to the calcium ionophore, A23187 in guinea-pig vas deferens, Br. J. Pharmacol. 71,581. Ito, S., Y. Nakazato and A. Ohga, 1978, The effect of the ionophores X-537A and A23187 on the noradrenaline output from peripheral adrenergic neurones in the presence of various divalent cations, Br. J. Pharmacol. 62, 91. Johansen, T., 1978, Mechanism of histamine release from rat mast cells induced by the ionophore A23187: effect of calcium and temperature, Br. J. Pharmacol. 63, 643. Johansen, T., 1979, Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187, Br. J. Pharmacol. 65, 103. Jori, A., A.P. Bentivoglio and S. Garattini, 1961, The mechanism of the local inflammatory reaction induced by compound 48/80 and dextran in rats, J. Pharm. Pharmacol. 13, 617. Knapp, H.R., O.O. Olez, J. Roberts, B.J. Sweetman, J.A. Oates and P.W. Reed, 1977, Ionophores stimulate prostaglandin and thromboxane biosynthesis, Proc. Nat. Acad. Sci. U.S.A. 74, 425 I. Lewis, A.J., D.J. Nelson and M.F. Sugrue, 1975, On the ability of prostaglandin E l and arachidonic acid to modulate experimentally induced oedema in the rat paw, Br. J. Pharmacol. 55, 51.
Lewis, G.P. and B.R. Whittle, 1977, The inhibition of histamine release from rat peritoneal mast cells by non-steroid anti-inflammatory drugs and its reversal by calcium, Br. J. Pharmacol. 61,229. Moncada, S., S.H. Ferreira and J.R. Vane, 1973, Prostaglandins, aspirin like drugs and the oedema of inflammation, Nature (London) 246, 217. Northover, B.J., 1977, Indomethacin--A calcium antagonist, Gen. Pharmacol. 8, 293. Parratt, J.R. and G.B. West, 1958, Inhibition by various substances of oedema formation in the hind paw of the rat induced by 5-hydroxytryptamine, histamine, dextran, eggwhite and compound 48/80, Br. J. Pharmacol. 13, 65. Piper, P.J. and J.P. Seale, 1978, Release of slow reacting substance from guinea-pig lung and human lung by calcium ionophore A23187, Br. J. Pharmacol. 63, 364 P. Pressman, B.C., 1973, Properties of ionophores with broad range cation selectivity, Fed. Proc. 32, 1698. Pressman, B.C., 1976, Biological applications of ionophores, Ann. Rev. Biochem. 45, 501. Scherrer, R.A., 1974, Aryl- and heteroarylcarboxylic acids, in: Anti-inflammatory Agents--Chemistry and Pharmacology, eds. R.A. Scherrer and M.W. Whitehouse (Academic Press, New York, London) Vol. l, p. 45. Spector, W.G. and D.A. Willoughby, 1968, in: The pharmacology of inflammation, eds. W.G. Spector and D.A. Willoughby (The English Universities Press Ltd., London) p. 66. Thomas, G., 1975, Pharmacological studies on non-steroidal anti-inflammatory compounds in the rat, Ph.D. Thesis, C.N.A.A.U.K. Thomas, G., 1980, Inhibition of calcium ionophore A23187 induced contraction of guinea-pig ileum by anti-inflammatory agents, J. Pharm. Pharmacol. 32, 141. Thomas, G. and G.B. West, 1973, Prostaglandins as regulators of bradykinin responses, J. Pharm. Pharmacol. 25, 747. Watson, E.L., 1978, Effect of ionophores A23187 and X-537A on vascular smooth muscle activity, European J. Pharmacol. 52, 171. Wedmore, C.V. and T.J. Williams, 1980, Evidence for two types of vascular permeability-increasing mediators: the direct action of histamine and bradykinin; the polymorphdependent action of C5a, leukotriene B4 and formyl tripeptide, Br. J. Pharmacol. 73, 209P. Williams, T.J., 1979, Prostaglandin E 2 and prostaglandin 12 and the vascular changes of inflammation, Br. J. Pharmacol. 65, 517. Williams, T.J. and J. Morley, 1973, Prostaglandins as potentiators of increased vascular permeability in inflammation, Nature (London) 246, 215. Williams, T.J. and M.J. Peck, 1977, Role of prostaglandin mediated vasodilatation in inflammation, Nature 270, 530.
35
MECHANISM OF IONOPHORE A23187 INDUCTION OF PLASMA PROTEIN LEAKAGE AND OF ITS INHIBITION BY INDOMETHACIN G. THOMAS
Laboratbrio de Tecnologia Farmac~utica, Universidade Federal da Paraiba, 58.000 Jo~o Pessoa, Paraiba, Brazil Received 8 September 1981, revised MS received 19 January 1982, accepted 1 March 1982
G. THOMAS, Mechanism of ionophore A23187 induction of plasma protein leakage and of its inhibition by indomethacin, European J. Pharmacol. 81 (1982) 35-42. Calcium ionophore A23187 produced a dose-dependent increase in plasma protein leakage on intradermal injection in rats. Studies with mepyramine and cyproheptadine indicated that histamine and 5-hydroxytryptamine (5-HT) partially contribute to the ionophore action and experiments with compound 48/80 supported these findings. Depletion of plasma kininogen levels with cellulose sulphate did not significantly inhibit the ionophore effect in rats suggesting that kinins are unimportant in the ionophore action. Orally administered indomethacin inhibited the ionophore response in a dose-dependent manner. The inhibition was not reversed by intradermally injected prostaglandin E 2 (PGE2) in doses up to 50 ng/site, suggesting that PGE 2 also may not be an important mediator. It is proposed that the ionophore produces plasma protein leakage by an indirect (through histamine and 5-HT release) and a direct action on vascular endothelial cells and that indomethacin antagonises both actions by inhibiting calcium transport processes. Ionophore A23187
Protein leakage
Mediators
Indomethacin
1. Introduction
Ionophore A23187 produces diverse pharmacological actions by its ability to transport divalent cations, preferentially calcium ions, across cell membranes (Pressman, 1973). Some of these recently reported actions include secretion of endocrine hormones (Pressman, 1976; Heisler, 1976), contraction of smooth muscle (Watson, 1978; Ishida and Shibata, 1980), stimulation of release of neurotransmitters (Casamenti et al., 1978; Ito et al., 1978), and promotion of axoplasmic transport of dopamine fl-hydroxylase (Esquerro et al., 1980). The ionophore was also shown to cause the release of histamine from rat mast cells (Foreman et al., 1973; Johansen, 1979), of SRS-A from human leucocytes (Conroy et al., 1976) and human and guinea-pig lungs (Piper and Seale, 1978), to stimulate the synthesis of thromboxane B2 and prostaglandins in rat polymorphonuclear leucocytes (PMNs) (Knapp et al., 1977) and the production of chemokinetic factors from human PMNs (Bray et al., 1980). 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
The objective of the present work was to determine whether ionophore A23187 has the ability to produce vascular permeability changes on intradermal injections into rats and if so, to study the mediators involved in this effect in comparison to the action of dextran, an agent known to produce its effect by releasing mainly 5-HT and histamine (Parratt and West, 1958). In studies conducted in vitro, the dextran response was also found to depend on calcium ions (Foreman and Mongar, 1972). Additional experiments were performed to observe whether indomethacin has antagonistic effects on the ionophore A23187 response, since other calcium-dependent effects of the ionophore, such as release of mast cell histamine and contraction of guinea-pig ileum, are inhibited by indomethacin (Lewis and Whittle, 1977; Thomas, 1980). Further, it has also been reported that indomethacin interferes with the participation of calcium ions in many other biological processes (Northover, 1977; Continenza et al., 1980).
36 2. Materials and methods
2.1. Vascular permeability studies with ionophore A23187 Male rats weighing 150-180g had their backs shaved 48 h before being used in experiments. Initially, pentobarbitone (40 m g / k g i.p.) was used as the anaesthetic agent. However, the ionophore was found to produce greater responses when ether was used and all subsequent experiments were therefore performed under ether anaesthesia. After anaesthetising the animals, Evans blue (20 mg/kg) was injected i.v. followed by i.d. injections of concentrations of the ionophore into the shaved areas, in dose volumes of 0.1 ml. A 2 m g / m l stock solution of the ionophore was prepared in ethanol, kept at - 18°C and diluted with saline as required. As each dose of the ionophore injected contained ethanol (the amount depending on the concentration of the ionophore), appropriate concentrations of ethanol in saline were also injected into different sites. Since maximal effects were produced between 30-45 rain after the ionophore applications, the rats were killed 45 min after the i.d. injections and the intensity of plasma protein leakage measured spectrophotometrically by estimating the amount of dye in each weal according to the method of Harada et al. (1971). The effects of the ionophore alone were obtained by subtracting the amount of dye exuded when diluted solutions of ethanol were injected from the appropriate responses to the ionophore. 2.2. Studies on mediators of ionophore A23187 response 2.2.1. Experiments with antagonistic drugs For the experiments with mepyramine, the dose of antagonist that inhibited (70-80%) the histamine response in the skin of rats was first established by injecting mepyramine i.p. 30 rain before the i.d. administration of a concentration of histamine that produced a submaximal response. This dose of mepyramine was administered i.p. into a group of 6 rats, followed 30 min later by i.d. injections of histamine, 5-HT, dextran and the ionophore. The doses of the agonists were such that they produced approximately similar skin
responses. Another group of 6 rats receiving saline i.p. served as control. Both groups received appropriate i.d. injections of ethanol and saline as well. The amount of dye which leaked at each site of injection was estimated as described earlier. Percent inhibition was calculated by comparing the responses (after subtracting the corresponding values for saline and ethanol injections) using the formula: [ ( R c - R t ) / R c ] X 100 where Rc and Rt are mean responses to agents in the saline- and mepyramine-treated groups respectively. When cyproheptadine was used as the antagonist, a concentration of cyproheptadine which inhibited the 5-HT response by more than 70% was administered i.p. 30 min before i.d. injections of histamine, 5-HT, dextran and the ionophore. Percent inhibition of responses for each agent was calculated as before. The effects of giving mepyramine and cyproheptadine simultaneously on the ionophore response were studied by giving both the antagonists in the doses used earlier and injecting the ionophore 30 min later. 2.2.2. Depletion studies Using compound 48/80, rats were depleted of their stores of histamine and 5-HT according to the schedule described by Di Rosa et al. (1971). Other rats receiving saline injections served as control. Both groups received i.d. injections of submaximal doses of dextran and the ionophore A23187 and the amount of dye exuded was estimated and the percent inhibition calculated as described before. Rats were depleted of plasma kininogen by repeated i.v. injections of cellulose sulphate as described by Di Rosa and Sorrentino (1970). This method lowers plasma kininogen levels by about 50%. The rats were used three hours after the last dose of cellulose sulphate for i.d. injections of dextran and ionophore. 2.3. Studies with indomethacin Giving indomethacin 12-24h before the ionophore injections was found to produce the most
37
marked inhibition. Therefore, doses of indomethacin were given orally to rats, 18 and 1 h before i.d. application of a selected submaximal dose of the ionophore. Three injections of ionophore and one injection of the vehicle were made in each animal. The effect of indomethacin on the protein exudation induced by the ionophore was determined by comparing the mean ionophore response in the control- and drug-treated groups. In a few experiments, doses of 10 or 50 ng/site of PGE 2 were also injected i.d. together with the ionophore to find whether the action of indomethacin (2 × 3.4 mg/kg) could be modified by externally applied PGE 2. Similar concentrations of PGE 2 alone were also injected to determine the effect of indomethacin on PGE 2 responses.
ing appropriate concentrations of ethanol in saline are also shown. Ionophore A23187 caused a concentration-dependent increase in plasma protein leakage, the largest dose of 21 #g/site producing an intense reaction spreading over a large area of the skin surrounding the site of injection. However, part of this response was due to the vehicle, a 12.5% solution of ethanol in saline which also produced marked dye leakage. Table 1 also demonstrates that the ionophore produced greater responses when ether was used as the anaesthetic agent in the animals. However, the results were significantly (P < 0.05) different only for 9 #g/site of the ionophore. Since injection of 9#g/site caused an easily measurable consistent response which was minimally affected by the vehicle (4.5% ethanol), this dose was selected for all subsequent experiments with the ionophore.
3. Results
3.1. Plasma protein leakage induced by the ionophore A23187 Table 1 shows the effects of i.d. injections of calcium ionophore A23187 on plasma protein leakage in rats under ether or under pentobarbitone anaesthesia. The results obtained after inject-
3.2. Mediators of ionophore A23187 response in the skin of rats 3.2.1. Experiments with antagonistic drugs The inhibitory effects of mepyramine and cyproheptadine on the ionophore response are shown in table2. A dose of 2.5 mg/kg i.p. of mepyramine significantly inhibited the responses
TABLE 1 Increase in plasma protein-bound dye leakage induced by i.d. injections of ionophore A23187 in ethanol and of 0.1 ml/site of appropriately diluted solutions of ethanol alone into groups of rats anaesthetized with either pentobarbitone or ether. Dye leakage (pg) was measured 45 min after the injections. Dose of ionophore and/~g of dye exuded
Concentration of ethanol and #g of dye exuded
Dose ~g
/~g of dye (A)"
% ethanol
Pentobarbitone
i.0 3.0 9.0 27.0
7.4±1.5" 10.1~1.1"* 19.0±1.3"** 30.0±4.1"*
0.5 1.5 4.5 12.5
2.6±0.1 2.0±0.1 3.5±0.2 7.6±1.1
4.8 8.1 15.5 22.4
Ether
1.0 3.0 9.0 27.0
9.9±2.0* 12.6±1.2"** 27.0±2.2*** 43.8±6.0**
0.5 1.5 4.5 12.5
2.5±0.1 3.0±0.3 3.5±0.3 11.0±2.5
7.4 9.6 23.5 32.8
Anaesthetic
/~g of dye (E)a
Dye exuded (#g) with ionophore alone (A--E)
Student's t-test: * P<0.025, ** P
38 TABLE 2 Antagonistic actions of mepyramine (2.5 m g / k g ) and cyproheptadine (2.5 m g / k g ) on plasma protein leakage induced by i.d. injections of histamine (10/~g), 5-HT (250 ng), dextran (9/~g) and ionophore A23187 (9 ~g) in groups of 6 rats. The mean amount of dye (/Lg/site) exuded with each agonist was measured 45 min after the i.d. injections. Agonist
Histamine 5-HT Dextran Ionophore A23187
/~g (mean ±S.E.M.) of dye e x u d e d / s i t e after pretreatment with
% inhibition of dye leakage with
Saline
Mepyramine
Mepyramine
Cyproheptadine
28.8 --+3.7 23.0--+3.5 30.0±3.6
6.8 + 1.1 *** 17.5± 1.5 NS 19.0+2.0 *
75.7 24.0 36.7
76.7 85.7 86.3
-
31.8--+4.0
25.2±4.0 ys
20.8
30.8
47.5
Cyproheptadine
Mepyramine + Cyproheptadine
6.7 ± 0.9 *** 3 . 3 ± 0 . 4 *** 4.1 +0.5 ***
-
22.0±2.2 *
16.7 + 1.2 **
Mepyramine + cyproheptadine
-
Student's t-test: * P < 0 . 0 5 , ** P<0.01, *** P<0.001. ys Not significant at the 95% probability level.
to histamine (P < 0.001) and dextran (P < 0.05). Mepyramine inhibited the responses to histamine, dextran, 5-HT and the ionophore by 75.7, 36.7, 24.0 and 20.8% respectively. Cyproheptadine in a dose of 2.5 mg/kg i.p. significantly antagonised the effects of histamine (P<0.005), 5-HT ( P < 0.001), dextran (P < 0.001) and the ionophore (P < 0.05). However, the degree of antagonism of the ionophore response (30.8%) was much less than that of the 5-HT, histamine and dextran responses (85.7, 76.7 and 86.3%, respectively). The combina-
tion of mepyramine and cyproheptadine also failed to inhibit the ionophore action to any great extent as the inhibition was only 47.5% suggesting that mechanisms other than histamine and 5-HT release contribute to the effect of the ionophore A23187 in the skin of rats.
3. 2.2. results of depletion studies The dextran response was significantly ( P < 0.001) reduced (79.1%) in compound 48/80pre-treated animals (table 3). Similar pre-treatment
TABLE 3 Effect of pretreatment of rats with compound 4 8 / 8 0 or cellulose sulphate on vascular permeability changes induced by intradermal injections of dextran (9 #g) and ionophore A23187 (9/lg) in groups in 6 rats. The mean amount (/~g/site) of dye exuded with each agent was determined 45 min after the i.d. injections. Test Agent
# g (mean - S . E . M . ) of dye e x u d e d / s i t e after pretreatment with Saline
Compound 48/80
Test I Dextran lonophore
25.3 ± 1.9 20.3±3.6
5.3 -4- 1.0 *** 12.2 + 1.1 *
Test H Dextran Ionophore
26.2 ± 1.6 22.0 ± 2.0
-
% inhibition of dye leakage with Cellulose sulphate
23.0 ± 3.0 NS 18.5 ± 2.1 NS
Student's t-test: * P<0 .0 5, *** P<0.001. NS Not significant at the 95% probability level.
Compound 48,/80
Cellulose sulphate
79.1 39.9
-
_ _
12.3 15.9
39 TABLE 4 Effect of graded doses of indomethacin on ionophore A23187-induced dye leakage in the skin of groups of 6 rats. Indomethacin was administered orally twice: 18 and 1 h before the injections of the ionophore. Dye leakage was estimated 45 min after the i.d. injections. Drug
Dose mg/kg p.o.
/,tg of dye exuded (mean ± S.E.M.)
Saline
10ml
20.5±2.6
1.25 2.5 5.0 10.0
16.5-+1.8 Ns 11.4±1.1" 8.3±1.9"* 3.3±1.0"**
% inhibition
EDs0 mg/kg
19.5 ~.3 59.5 83.9
3.4
Indomethacin
Student's t-test: * P < 0 . 0 5 , ** P<0.01, *** P<0.001. NS Not significant at the 95% probability level.
3.3. Inhibitory studies with indomethacin
with compound 48/80 also inhibited the ionophore effect in a significant (P < 0.05) manner, but the inhibition (39.1%) was much less than that obtained with the dextran skin reaction. Table 3 also demonstrates that previous depletion of plasma kinins by cellulose sulphate did not significantly reduce either the dextran- or the ionophore-induced protein leakage in rats. The dextran response was inhibited by 12.3% and the ionophore response by 15.9% when compared with saline-treated control animals.
Various doses of indomethacin, each given orally twice, i.e. 18 and 1 h before the i.d. injections of the ionophore, produced dose-related inhibition of the ionophore-induced dye leakage in rats (table 4). The inhibition was statistically significant for the 2.5 (P < 0.05), 5.0 (P < 0.01) and 10 mg/kg (P < 0.001) doses and the approximate EDs0 value calculated from the dose-response curve was 3.4 mg/kg.
TABLE 5 Experiment I demonstrates the inability of PGE 2 (10 n g / s i t e i.d.) to potentiate the increased dye leakage produced by ionophore A23187 (9 # g/site) and also the failure of this dose of PGE 2 to reverse the inhibitory activity of indomethacin (2 × 3.4 m g / k g p.o.) on the ionophore response. Similar results obtained with 50 n g / s i t e of PGE 2 are given in experiment II. Dye exudation was measured 45 min after i.d. injection in groups of 6 rats. Agent injected i.d. and dose
Experiment I P G E 2 10 ng Ionophore 9/~g Ionophore 9/~ g + PGE 2 10 ng Experiment 11 P G E 2 50 ng lonophore 9/~g Ionophore 9 / ~ g + P G E 2 50 ng
/~g (mean ±S.E.M.) of dye exuded after treatment with
% inhibition with Indometbacin
Saline
Indomethacin
2.6--+0.6 22.9±2.1 23.1 ± 2.4
2.1 ± 0 . 4 NS 12.0-+0.8 ** 11.6 ± 0.5 **
19.2 47.6 49.8
5.0± 1.6 21.4±2.0 25.0±2.6
4.1 ± 1.2 NS 12.6± 1.2 * 16.0± 1.0 *
18.0 40.2 36.0
Student's t-test: * P<0.05, ** P<0.01. Ns Not significant at the 95% probability level.
40 The effects of externally applied PGE 2 on the ionophore response are shown in table 5. On i.d. injection, PGE 2 in doses of either 10 or 50 ng/site, produced only minimal dye leakage which was not significantly ( P < 0 . 1 ) affected by indomethacin (2 × 3.4 mg/kg). In the above doses PGE 2 also failed to potentiate the ionophore response when injected together with this agent. The table also shows that PGE 2 did not modify the inhibitory activity of indomethacin on the ionophore-induced dye leakage. Indomethacin produced inhibition of 47.6 and 49.8% in the absence of PGE 2 or with PGE2, respectively. At the larger indomethacin dose (50 ng/site), inhibition was 40.2 and 36.0% in the absence or presence of PGE 2, respectively. In all these cases, inhibition was statistically significant (P < 0.5 or below).
4. Discussion
Intradermal injections of ionophore A23187 produced dose-related increases in plasma protein leakage in the skin of rats. Since doses of 9/xg/site of both dextran and ionophore A23187 induced responses of approximately similar intensity it is possible that the ionophore was equipotent with dextran in producing vascular permeability changes. This can only be confirmed by comparing the dose-response curves of these two agents. For investigating the mechanism of action of A23187, dextran was selected as the reference agent as the mediation of dextran reaction is well known (Parrett and West, 1958) and further, a number of studies comparing dextran and compound A23187 have been reported (Garland and Mongar, 1976; Foreman et al., 1977). Experiments in separate groups of rats with the specific antagonists mepyramine and cyproheptadine, indicated that when compared to dextran, neither histamine nor 5-HT has a major role in the mediation of the ionophore response in the skin of rats. These resuits are further supported by the fact that less than 50% inhibition of the ionophore response was obtained when this agent was injected into rats given both mepyramine and cyproheptadine previously. The doses of antagonists were such that these agents inhibited the responses to specific
agonists by more than 70%. In addition, the 2.5 m g / k g dose of cyproheptadine alone was sufficient to produce almost complete inhibition of the dextran response. Pre-treatment of rats with compound 48/80 to deplete the stores of histamine and 5-HT in the body suppressed the ionophore response far less than the dextran response, providing further evidence for the relatively minor roles of histamine and 5-HT in the ionophore response. Treatment of rats with cellulose sulphate, a kininogen-depleting agent, failed to inhibit significantly either the dextran- or A23187-induced plasma protein exudation. These results indicate that the action of kinins is not important in this respect even though in other experimental models, kinins have been implicated in the mediation of the dextran response (Garcia Leme et al., 1967; Di Rosa and Sorrentino, 1970). In the present tests PGE 2 did not potentiate the ionophore action even though PGEs have shown such effects in several models (Moncada et al., 1973; Williams and Morley, 1973; Lewis et al., 1975). However, PGE l was also reported to have no potentiating effect on histamine, 5-HT, dextran and compound 48,/80 responses (Jori et al., 1961; Lewis et al., 1975). Lewis et al. (1975) attempted to explain the potentiating effect of PGE 1 and PGE 2 in carrageenan and kaolin-induced rat paw oedema by their ability to act synergistically with kinins released during these responses. Thomas and West (1973) also found that PGE 1 potentiated the bradykinin response while having no effect on the histamine, 5-HT and dextran responses in rat skin. Thus the failure of PGE 2 to modify the ionophore response may be explained partially by the minor role of kinins in this reaction. It has been proposed (Williams and Peck, 1977; Williams, 1979) that an agent produces maximum plasma protein exudation if it has a vasodilatory action in addition to its vascular permeability-increasing property. For example, Wedmore and Williams (1980) demonstrated that three leucotactic agents with minimal intrinsic vasodilatory action produced significant plasma exudation only when PGE z was injected along with these agents. Ionophore A23187 may possess potent vasodilatory properties, in which case the addition of
41
another vasodilator may have no further effect; such an explanation could perhaps also account for the lack of effect of PGE 2 in these experiments. Prostaglandin responses are highly variable in the rat (Freeman and West, 1972) and the intensity of the reaction produced by PGEs even in the maximal doses of 2.5 #g/site is far less than that of the response to compound A23187 and mediators of inflammation (Thomas, 1975). In the present studies, the indomethacin-induced inhibition of the ionophore response was also not reversed by PGE 2 in doses upto 50 ng/site. All these results suggest that PGs may not contribute to the ionophore response in the skin of rats. Further experiments are required to determine the true role of PGs, as calcium ionophores have been shown (Knapp et al., 1977) to stimulate the synthesis of PGs. It is probable that ionophore A23187 causes increased vascular permeability to plasma proteins by a direct action on the vascular endothelial cell as well. Thus, by raising the concentration of intracellular calcium, the ionophore may act on the contractile elements of the cells to produce interendothelial gaps through which plasma proteins may leak. It is proposed that, unlike dextran which acts indirectly by releasing chemical mediators, ionophore A23187 has an additional direct effect in causing plasma protein leakage. Indomethacin produced a dose-dependent inhibition of plasma protein exudation induced by the ionophore. In general non-steroid anti-inflammatory drugs antagonise the vascular permeability changes induced by mediators of inflammation in high doses only, for example, even a dose of 10 mg/kg i.v. of indomethacin failed to inhibited bradykinin-induced dye leakage in rats (Spector and Willoughby, 1968). However, the EDs0 value of 2 × 3.4 mg/kg obtained for indomethacin in the present experiments is close to the EDs0 values of 1.4-2.0 and 2.2-6.5 mg/kg p.o. found in guinea-pig u.v. erythema and rat carrageenan oedema respectively (Scherrer, 1974). Since both indirect and direct actions of the ionophore A23187 depend on transport of extracellular calcium into target cells (Pressman, 1973; Garland and Mongar, 1976; Johansen, 1978; Desmedt and
Hainaut, 1979), the present results support the findings (Famey and Whitehouse, 1976; Northover, 1977; Lewis and Whittle, 1977; Thomas, 1980; Continenza et al., 1980) that indomethacin and other non-steroid anti-inflammatory drugs have effects on calcium transport process in biological membranes. However, the importance of this action in relation to the anti-inflammatory effect of such compounds has yet to be assessed.
Acknowledgment I wish to thank Prof. Delby F. de Medeiros, Director of Laboratrrio de Tecnologia Farmac~utica for his encouragement, Eli Lilly & Co for the gift of ionophore A23187 and Mr Jos~ C. Duarte and Mrs Clebia G. de Oliveira for excellent technical assistance.
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