PROSTAGLANDINS
SELECTIVE INHIBITION OF PROSTAGLANDIN BIOSYNTHESIS BY GOLD SALTS AND PHENYLBUTAZONE
K J Stone, S J Mather and P P Gibson
Department of Biochemistry, Roche Products Ltd. Broadwater Road, Welwyn Garden City, Herts. England.
SUMMARY
Gold salts and phenylbutazone selectively inhibit the synthesis of PGF2, and PGE2.respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE2 and PGF2, syntheses equally. It is postulated that selective inhibitors may have a different mode of action _in vivo and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.
INTRODUCTION One of the most fascinating aspects of the biology of prostaglandins is the way individual tissues of the body may respond quite differently to different prostaglandins. -Forexample PGE2 increases vascular skin permeability (l), relaxes bronchiolar tissues (2,3), is a vasodilator (4) and promotes natriuresis and increased urine flow (5). On the other hand PGFa antagonises the vascular permeability caused by PGE2 (6), constricts bronchiolar tissue (3), is pressor in dons and rats (7) and has little effect on sodium and water excretion (8):' Such observations suggest that an imbalance of prostaglandins in a particular tissue could result in an abnormal regulation of, for example, blood pressure control and disorders associated with inflammation. Some support for this idea has been provided by the observation that the ratio of PGE2 to PGF2, is raised in carrageenin-induced pleurisy
AUGUST
1975
VOL. 10 NO. 2
241
PROSTAGLANDINS
and carrageenin peritonitis (9). In situations of prostaglandin imbalance it would clearly be a major advance if the relative levels of PGE2 and PGF2a could be manipulated by drug therapy. The biosyntheses of PGE2 and PGF2, follow a common pathway up to prostaglandin endoperoxide and isotopic evidence suggests that the subseq'lentreactions of endoperoxide isomerase and endoperoxide reductase compete for the same pool of substrate (10). In this paper we report the ability of a number of synthetase inhibitors to selectively affect these subsequent reactions and suggest that the resulting redirection of synthesis could explain the variable response of patients to non-steroidal anti-inflammatory drugs.
METHODS -Unlabelled arythidonic acid (Nu-Check-PREP. Elysian, Minn. USA) and [l- Cl-arachidonic acid (specific activity 54 mCi/mmol, Radiochemical Centre, Amersham) were each dissolved in an anti-oxidant solution containing 0.01 ml benzyl alcohol, 4 mg disodium EDTA, 2 ng propyl gallate, 2 mg butvlatedhvdroxstoluene and 40 mn sodium metabisulnhite ner ml of solu%_on adjusted to pH 9-g with NaOH. The conceniration of unlabelled arachidonate was 5 mg/ml and the concentration of radiolabelled arachidonate was 5 pCi/ml. Each solution was stored under nitrogen at - 20". It was essential to pre-silanise all glassware which came into contact with arachidonic acid. Prostaglandin synthetase was prepared from the frozen seminal vesicles of sheer,b-v nrecinitation with citric acid followed by treatment wi%h acetone-and pentane essentially as described bv Wallach L?C Daniels ill). Before use the enzyme powder was homogenised gently with 30 volumes of O.lM Tris-HCl buffer, pH 8.4, in a glass homogeniser. Ten volumes of 25 mM adrenaline were added to give a solution containing 12 mg protein/ml as measured according to Lowry -et al (12). This solution, containing 6.25 mM adrenaline, was preincubated at 25" for 5 min before use. Before any series of experiments the enzyme reaction mixture was titrated with Cu++ to yield an equimolar ratio of Although different batches of enzyme powder PGE2 : PGF%required di ferent amounts of CL@, the titration value remained constant for any one preparation. Measurements of enzyme activity were performed in 15 ml silanised conical centrifuge tubes at 25". Reaction mixtures consisted of reduced glutathione (1.1 x 10_3M), copper sulphate (2.5 x lo-5M), O.lM Tris-HCl buffer DH 8.4, ?&o.ibitor dissolved in C-05 ml x&star-ethanol and enzyme/adrenaline solution
242
AUGUST
1975 VOL. 10 NO. 2
PROSTAGLANDINS
(0.02 ml). The total volume was 0.48 ml. This mixture was pre-incubated at 25" for 5 min and the reaction was initiated with 0.02 ml substrate solution containing 0.05 @i, 5 pg arachidonate. After 5 min incubation at 25" the reaction was terminated by the addition of O-1 ml of a 2 M citric acid, 3.3 E saline solution. Analar ethyl acetate-(O-15 ml) was added and the mixture vortexed and centrifuged. A portion (O-02 ml) of the upper organic phase was subjected to thin-layer chromatography on silica gel G-25 (Macherey-Nagel & Co) using 1% formic acid in ethyl acetate as the solvent system. Following chromatography, chromatograms were dried and scanned with a Panax radiochromatogram scanner to orovide a visual record of the distribution of radioactivity-and to confirm the co-mobility of radioactivity with authentic Good separations of arachidongte markers of PGE2 and PGF (RF, o-931, PGE (RFyoe% PGF~ (RF, 0.25) and ll-dehydroPGF2, (RF, o-83$ were obtained. Radioactive areas were scraped from the cbromatogram directly into scintillation vials and assaved bv liauid scintiliation spectrometry. Results have been Icalculatedfrom the amount of radioactivity on a particular area of the plate expressed as a percentage of the total radioactivity recovered after chromatography. Percentage inhibition and percentage stimulation are calculated with reference to the control incubations which contained no inhibitor. Inhibitors were meclofenamic acid (Parke-Davis), indomethacin (Sigma), flurbiprofen (Boots), ibuprofen (Boots), ketoprofen (May & Baker), phenylbutazone (Geigy), acetylsalic late (Aspirin, BDH), hydroxychloroquine sulphate (WinthropB , 5,8,11,14eicosatetraynoic acid (Ro 03-1428, Roche), sodium aurothiomalate (Aldrich) and aurothioglucose (Sigma).
RESJJLTS AND DISCUSSION Detailed chromatographic analyses of products synthesised from Cl-14C]-arachidonate by prostaglandin synthetase of sheep seminal vesicles have shown that adrenaline increases the total metabolism of substrate but does not affect the distribution of products. Reduced glutathione (GSH), however, specifically channels the synthesis into PGE2 production whilst the addition of Cu++ to incubations containing adrenaline and GSH stimulates the formation of PGF2o at the expense of PGE2 (13). In such incubations PGE and PGF2o together represent 8@h of the total products wi.zh hydroxy fatty-acids and ll-dehydroPGF2o representing 6% and 14% respectively. All inhibitors were tested under conditions where the enzyme, substrate and cofactor levels had been adjusted to give 6@b utilisation of substrate with PGE2 and PGF2, being formed in approximately equal amounts in the control incuba-
AUGUST
1975
VOL. 10 NO. 2
243
PROSTAGLANDINS
tions. Varied concentrations of inhibitor were used and the inhibition or stimulation of PGE2 and PGF& production was measured. Different batches of enzyme, which required different concentrations of Cu+ to produce equimolar amounts of PGE2 could not be distinguished by their response to a %~%% inhibitor. The results. as shown in Table 1 and Fig. 1, indicate that the inhibitors can be grouped into three types. Non-selective inhibitors This group contains the fenamates, a-methylacetic acid derivatives, indomethacin and substrate analogues, all of which inhibit PGE2 and PGFza production equally. It is presumed, therefore, that these compounds have a site of action before the endoperoxide stage and most probably at, the level of the dioxygenase reaction. Such a site of aotion has previous1 been suggested for the acetylenic substrate analogues (143 and indomethacin (15,16). InhiBition of this initial reaction must reduce the amount o'f substrate available for endoperoxide reductase and endoperoxide isomerase. In bovine seminal vesicles these two enzymes have different kinetic characteristics and a change in the PGEz/PGFzo ratio occurs as the substrate level is changed (16). The enzyme from sheep seminal vesicles has quite different properties and no change in PGE2/PGFza ratio is seen when the arachidonic acid concentration is varied (13), nor when the reaction is partially inhibited with indomethacin (Fig. 1). F2-selective
inhibitors
Inhibition of PGE2 synthesis by phenylbutazone is accomoanied at lower concentrations bv a stimulation of PGF% synthisis so that the net effect on total prostaglandin synthesis is negligible. These results suggest that phenylbutazone selectively inactivates the endoperoxide isomerase although other explanations such as effects on the cofactor requirements cannot be ruled out. Under these conditions when endoperoxide isomerase is selectively blocked, endoperoxide reductase is essentially the only metabolic route remaining and thus an increase in PGFza is not unexpected. When bovine seminal vesicles are used as the enzyme source, phenylbutazone inhibits both PGE2 and PGF but has no effect on ll-dehvdroPGFom and malondialdehvde (16 3 . This either illustrates a fu?xher difference be'tween the two enzymes or might indicate that Cu++ (which is not added to the bovine system to generate PGF&)‘interacts with phenylbutazone to produce the selective inhibition of PGE2 synthesis. A selective inhibition of PGE synthesis by aspirin has et al (17) who utilised recently been reported by Horodnia.Z-bovine seminal vesicle microsomes incubated with arachidonate and L-adrenaline bitartrate. We have frequently observed a similar differential inhibition by aspirin when GSH and CU++
244
AUGUST
1975 VOL. 10 NO. 2
PROSTAGLANDINS
are omitted from the incubation (Fig. 2A). In the presence of these cofactors, however, such a selective inhibition (or stimulation) is not apparent (Fig. 2B). The significance of this result is unclear as we have found that incubations performed in the absence of GSH and Cu++ are sometimes difficult to interpret because of the multiplicity of products. It may be that in the absence of cofactors a less rigid competition for the endoperoxide occurs and the results reflect modifications of a non-enzymic re-arrangement of this substrate (18). The effect of pre-incubating CuC12 with a crude homogenate of sheep vesicular tissue for a minimum of 15 min accelerates a time-dependent inactivation of "PGE2-synthetase" with a stimulation of "PGF2a-synthetase" (19). Following such an activation/inactivation process few anti-inflammatory compounds inhibited "PGF2a-synthetase" although some inhibition of PGEq-synthesis was reoorted (19). Under our reaction conditions there were apparently no time-dependent effects of copper, and maximum re-direction of synthesis occurred in less than 5 min. Furthermore, most anti-inflammatory drugs inhibited PGF2a synthesis and PGE2 synthesis equally. PGFh-specific -
inhibitors
Sodium aurothiomalate and aurothioglucose dramatically inhibited PGF2a synthesis and stimulated PGE2 synthesis. " This is the oowosite effect to that created bv dithiols in the presence of Cu++ (20). It seems likely, %herefore, that the combination of dithiols or reduced glutatbione with Cu++ maintains a reduced thiol group, at the active site of prostaglandin endoperoxide reductase (21), which gold salts interact with and inactivate (22). Bydroxychloroquine sulphate had a specificity which was less pronounced than that of the gold salts. This effect has also been seen with chloroquine and prostaglandin biosynthesis in rat skin homogenates (23). It is curious that these drugs should have the effect of stimulating PGE2 synthesis whilstinhibiting PGFa synthesis as one would have thought that, as increased PGE to PGF ratios can be correlated with certain types of inflammation, any re-direction of synthesis towards PGE would exacerbate inflammation. It is interesting that chloroquine has other properties, possibly related to its ability to destabilise membranes (24), which apparently contradict the desired mode of action for an anti-inflammatory drug, e.g. it potentiates the oedema in adjuvant arthritis (25) and stimulates the breakdown of cartilage grown in tissue culture (26). Superficially these results are not in accord with those of Ziboh et al (27) who found that aurothiomalate and chloroauine inhzizd the oxygen uwtake of a ohenol-stimulated acetone-pentane powder"of sheep seminal-vesicles by 50"/0 when the enzyme was incubated with arachidonate in the absence of
AUGUST
1975
VOL. 10 NO. 2
245
PRO’STAGLANDINS
glutathione and copper. It appears, therefore, that one or more of these cofactors, which channel the synthesis towards PGE and PGF, might also protect the dioxygenase component of the synthetase complex from inhibition by these compounds. Inconclusion, the results provided by this study show that under the same reaction conditions different anti-inflammatory drugs may either block the synthesis of both PGE2 and PGF2 or selectively inhibit the synthesis of one. If pros? aglandin synthetase in man is similarly affected the difference in response of some patients to either indomethacin or phenylbutazone could be attributable to such different modes of action. The totally unexpected observation that gold salts are completely specific in inhibiting PGF& synthesis whilst stimulating PGE2 synthesis might explain why gold salts are found useful in clinical situations where conventional antiinflammatory drugs are ineffective. Possibly in these situations the "anti-rheumatic" effect of PGE, which reduces lysosomal enzyme release (28) and inhibits experimentally induced polyarthritis in rats (29), may operate. It is tempting to speculate that an important factor which determines the drug of choice for a particular patient is the endogenous ratio of PGE to PGF required for normal function. Patients whose normal function reauires a relatively high PGF level (compared to PGE) would be expected to respond" better to phenylbutazone than to either gold salts or indomethacin and vice versa. However, when considering inflammation, such patients appear to be in a minority and total blockade of all prostaglandin biosynthesis by aspirin or indomethacin is generally beneficial. On the other hand, whilst administered prostaglandin may have a potent effect on blood pressure, inhibition of prostaglandin-biosynthesis by aspirin or indomethacin has little effect. This would suggest that the net effect on blood pressure of opposing acTions of different prostaglandins is insignificant. Thus a drug which acted like the gold salts and increased the ratio of PGE to PGF could be of value in controlling hypertension. REFERENCES 1.
BergstrBm, S., Duner, H., Van Euler, U.S., Pernow, B. & Acta Physiol. Stand. 45, 145-151 Sjovall, J. (1959 > Kaley, G. & Weiner, R. 180, 338-350
(1971)
Crunkhorn, P. & Willis, A.L. 5, 49
Ann. N.Y. Acad. Sci.
(1971)
AUGUST
Brit. J. Pharmacol.
1975 VOL. 10 NO. 2
PROSTAGLANDINS
2.
Rosenthale, M.E., Dervinis, A., Begany, A.J., Lapidos, M. Experientia 26, 1.119-1121 & Gluckman, M.I. (1970) Cuthbert, M.F.
3.
James, G.W.Z. Puglisi, L. p. 219-227,
(1969) (1969)
Brit. Med. J.
",
723
J. Pharm. Pharmac.
2,
379-386
in "Advances in the Biosciences 9” (1973) Pergamon Press, Ed. Bergstrdm
4.
Horton, E.W.
5.
McGiff, J.C. & Nasjletti, A. (;~',~~mi~p~;~sta&andins . and Cyclic AMP", p. 119-151, 9 Kahns & Lands
6.
Crunkhorn, P. and Willis, A.L. Pharmacol. 5, 507-512
7.
Du Charme, D.W. & Weeks, J.R. (1967) in "Nobel Symposium 2",p.173-181, Interscience, Ed. BergstrBm & Samuelsson
8.
Lonigro, A.J., Terragno, N.A., Malik, K.U. & McGiff, J-C. (1973) Prostaglandins 2, 595-606
9.
Giroud, J-P. (1973) Abs. Intern. Meeting on "Future Trends in Inflammation", Verona
10.
Wlodawer, P. & Samuelsson, B. 248 5673-5678 -'
(1973)
J. Biol. Chem.
11.
Wallach, D.P. & Daniels, E.G. Acta 231, 445-457
(1971)
Biochim. Biophys.
12.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (19
SELECTIVE INHIBITION OF PROSTAGLANDIN BIOSYNTHESIS BY GOLD SALTS AND PHENYLBUTAZONE
K J Stone, S J Mather and P P Gibson
Department of Biochemistry, Roche Products Ltd. Broadwater Road, Welwyn Garden City, Herts. England.
SUMMARY
Gold salts and phenylbutazone selectively inhibit the synthesis of PGF2, and PGE2.respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE2 and PGF2, syntheses equally. It is postulated that selective inhibitors may have a different mode of action _in vivo and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.
INTRODUCTION One of the most fascinating aspects of the biology of prostaglandins is the way individual tissues of the body may respond quite differently to different prostaglandins. -Forexample PGE2 increases vascular skin permeability (l), relaxes bronchiolar tissues (2,3), is a vasodilator (4) and promotes natriuresis and increased urine flow (5). On the other hand PGFa antagonises the vascular permeability caused by PGE2 (6), constricts bronchiolar tissue (3), is pressor in dons and rats (7) and has little effect on sodium and water excretion (8):' Such observations suggest that an imbalance of prostaglandins in a particular tissue could result in an abnormal regulation of, for example, blood pressure control and disorders associated with inflammation. Some support for this idea has been provided by the observation that the ratio of PGE2 to PGF2, is raised in carrageenin-induced pleurisy
AUGUST
1975
VOL. 10 NO. 2
241
PROSTAGLANDINS
and carrageenin peritonitis (9). In situations of prostaglandin imbalance it would clearly be a major advance if the relative levels of PGE2 and PGF2a could be manipulated by drug therapy. The biosyntheses of PGE2 and PGF2, follow a common pathway up to prostaglandin endoperoxide and isotopic evidence suggests that the subseq'lentreactions of endoperoxide isomerase and endoperoxide reductase compete for the same pool of substrate (10). In this paper we report the ability of a number of synthetase inhibitors to selectively affect these subsequent reactions and suggest that the resulting redirection of synthesis could explain the variable response of patients to non-steroidal anti-inflammatory drugs.
METHODS -Unlabelled arythidonic acid (Nu-Check-PREP. Elysian, Minn. USA) and [l- Cl-arachidonic acid (specific activity 54 mCi/mmol, Radiochemical Centre, Amersham) were each dissolved in an anti-oxidant solution containing 0.01 ml benzyl alcohol, 4 mg disodium EDTA, 2 ng propyl gallate, 2 mg butvlatedhvdroxstoluene and 40 mn sodium metabisulnhite ner ml of solu%_on adjusted to pH 9-g with NaOH. The conceniration of unlabelled arachidonate was 5 mg/ml and the concentration of radiolabelled arachidonate was 5 pCi/ml. Each solution was stored under nitrogen at - 20". It was essential to pre-silanise all glassware which came into contact with arachidonic acid. Prostaglandin synthetase was prepared from the frozen seminal vesicles of sheer,b-v nrecinitation with citric acid followed by treatment wi%h acetone-and pentane essentially as described bv Wallach L?C Daniels ill). Before use the enzyme powder was homogenised gently with 30 volumes of O.lM Tris-HCl buffer, pH 8.4, in a glass homogeniser. Ten volumes of 25 mM adrenaline were added to give a solution containing 12 mg protein/ml as measured according to Lowry -et al (12). This solution, containing 6.25 mM adrenaline, was preincubated at 25" for 5 min before use. Before any series of experiments the enzyme reaction mixture was titrated with Cu++ to yield an equimolar ratio of Although different batches of enzyme powder PGE2 : PGF%required di ferent amounts of CL@, the titration value remained constant for any one preparation. Measurements of enzyme activity were performed in 15 ml silanised conical centrifuge tubes at 25". Reaction mixtures consisted of reduced glutathione (1.1 x 10_3M), copper sulphate (2.5 x lo-5M), O.lM Tris-HCl buffer DH 8.4, ?&o.ibitor dissolved in C-05 ml x&star-ethanol and enzyme/adrenaline solution
242
AUGUST
1975 VOL. 10 NO. 2
PROSTAGLANDINS
(0.02 ml). The total volume was 0.48 ml. This mixture was pre-incubated at 25" for 5 min and the reaction was initiated with 0.02 ml substrate solution containing 0.05 @i, 5 pg arachidonate. After 5 min incubation at 25" the reaction was terminated by the addition of O-1 ml of a 2 M citric acid, 3.3 E saline solution. Analar ethyl acetate-(O-15 ml) was added and the mixture vortexed and centrifuged. A portion (O-02 ml) of the upper organic phase was subjected to thin-layer chromatography on silica gel G-25 (Macherey-Nagel & Co) using 1% formic acid in ethyl acetate as the solvent system. Following chromatography, chromatograms were dried and scanned with a Panax radiochromatogram scanner to orovide a visual record of the distribution of radioactivity-and to confirm the co-mobility of radioactivity with authentic Good separations of arachidongte markers of PGE2 and PGF (RF, o-931, PGE (RFyoe% PGF~ (RF, 0.25) and ll-dehydroPGF2, (RF, o-83$ were obtained. Radioactive areas were scraped from the cbromatogram directly into scintillation vials and assaved bv liauid scintiliation spectrometry. Results have been Icalculatedfrom the amount of radioactivity on a particular area of the plate expressed as a percentage of the total radioactivity recovered after chromatography. Percentage inhibition and percentage stimulation are calculated with reference to the control incubations which contained no inhibitor. Inhibitors were meclofenamic acid (Parke-Davis), indomethacin (Sigma), flurbiprofen (Boots), ibuprofen (Boots), ketoprofen (May & Baker), phenylbutazone (Geigy), acetylsalic late (Aspirin, BDH), hydroxychloroquine sulphate (WinthropB , 5,8,11,14eicosatetraynoic acid (Ro 03-1428, Roche), sodium aurothiomalate (Aldrich) and aurothioglucose (Sigma).
RESJJLTS AND DISCUSSION Detailed chromatographic analyses of products synthesised from Cl-14C]-arachidonate by prostaglandin synthetase of sheep seminal vesicles have shown that adrenaline increases the total metabolism of substrate but does not affect the distribution of products. Reduced glutathione (GSH), however, specifically channels the synthesis into PGE2 production whilst the addition of Cu++ to incubations containing adrenaline and GSH stimulates the formation of PGF2o at the expense of PGE2 (13). In such incubations PGE and PGF2o together represent 8@h of the total products wi.zh hydroxy fatty-acids and ll-dehydroPGF2o representing 6% and 14% respectively. All inhibitors were tested under conditions where the enzyme, substrate and cofactor levels had been adjusted to give 6@b utilisation of substrate with PGE2 and PGF2, being formed in approximately equal amounts in the control incuba-
AUGUST
1975
VOL. 10 NO. 2
243
PROSTAGLANDINS
tions. Varied concentrations of inhibitor were used and the inhibition or stimulation of PGE2 and PGF& production was measured. Different batches of enzyme, which required different concentrations of Cu+ to produce equimolar amounts of PGE2 could not be distinguished by their response to a %~%% inhibitor. The results. as shown in Table 1 and Fig. 1, indicate that the inhibitors can be grouped into three types. Non-selective inhibitors This group contains the fenamates, a-methylacetic acid derivatives, indomethacin and substrate analogues, all of which inhibit PGE2 and PGFza production equally. It is presumed, therefore, that these compounds have a site of action before the endoperoxide stage and most probably at, the level of the dioxygenase reaction. Such a site of aotion has previous1 been suggested for the acetylenic substrate analogues (143 and indomethacin (15,16). InhiBition of this initial reaction must reduce the amount o'f substrate available for endoperoxide reductase and endoperoxide isomerase. In bovine seminal vesicles these two enzymes have different kinetic characteristics and a change in the PGEz/PGFzo ratio occurs as the substrate level is changed (16). The enzyme from sheep seminal vesicles has quite different properties and no change in PGE2/PGFza ratio is seen when the arachidonic acid concentration is varied (13), nor when the reaction is partially inhibited with indomethacin (Fig. 1). F2-selective
inhibitors
Inhibition of PGE2 synthesis by phenylbutazone is accomoanied at lower concentrations bv a stimulation of PGF% synthisis so that the net effect on total prostaglandin synthesis is negligible. These results suggest that phenylbutazone selectively inactivates the endoperoxide isomerase although other explanations such as effects on the cofactor requirements cannot be ruled out. Under these conditions when endoperoxide isomerase is selectively blocked, endoperoxide reductase is essentially the only metabolic route remaining and thus an increase in PGFza is not unexpected. When bovine seminal vesicles are used as the enzyme source, phenylbutazone inhibits both PGE2 and PGF but has no effect on ll-dehvdroPGFom and malondialdehvde (16 3 . This either illustrates a fu?xher difference be'tween the two enzymes or might indicate that Cu++ (which is not added to the bovine system to generate PGF&)‘interacts with phenylbutazone to produce the selective inhibition of PGE2 synthesis. A selective inhibition of PGE synthesis by aspirin has et al (17) who utilised recently been reported by Horodnia.Z-bovine seminal vesicle microsomes incubated with arachidonate and L-adrenaline bitartrate. We have frequently observed a similar differential inhibition by aspirin when GSH and CU++
244
AUGUST
1975 VOL. 10 NO. 2
PROSTAGLANDINS
are omitted from the incubation (Fig. 2A). In the presence of these cofactors, however, such a selective inhibition (or stimulation) is not apparent (Fig. 2B). The significance of this result is unclear as we have found that incubations performed in the absence of GSH and Cu++ are sometimes difficult to interpret because of the multiplicity of products. It may be that in the absence of cofactors a less rigid competition for the endoperoxide occurs and the results reflect modifications of a non-enzymic re-arrangement of this substrate (18). The effect of pre-incubating CuC12 with a crude homogenate of sheep vesicular tissue for a minimum of 15 min accelerates a time-dependent inactivation of "PGE2-synthetase" with a stimulation of "PGF2a-synthetase" (19). Following such an activation/inactivation process few anti-inflammatory compounds inhibited "PGF2a-synthetase" although some inhibition of PGEq-synthesis was reoorted (19). Under our reaction conditions there were apparently no time-dependent effects of copper, and maximum re-direction of synthesis occurred in less than 5 min. Furthermore, most anti-inflammatory drugs inhibited PGF2a synthesis and PGE2 synthesis equally. PGFh-specific -
inhibitors
Sodium aurothiomalate and aurothioglucose dramatically inhibited PGF2a synthesis and stimulated PGE2 synthesis. " This is the oowosite effect to that created bv dithiols in the presence of Cu++ (20). It seems likely, %herefore, that the combination of dithiols or reduced glutatbione with Cu++ maintains a reduced thiol group, at the active site of prostaglandin endoperoxide reductase (21), which gold salts interact with and inactivate (22). Bydroxychloroquine sulphate had a specificity which was less pronounced than that of the gold salts. This effect has also been seen with chloroquine and prostaglandin biosynthesis in rat skin homogenates (23). It is curious that these drugs should have the effect of stimulating PGE2 synthesis whilstinhibiting PGFa synthesis as one would have thought that, as increased PGE to PGF ratios can be correlated with certain types of inflammation, any re-direction of synthesis towards PGE would exacerbate inflammation. It is interesting that chloroquine has other properties, possibly related to its ability to destabilise membranes (24), which apparently contradict the desired mode of action for an anti-inflammatory drug, e.g. it potentiates the oedema in adjuvant arthritis (25) and stimulates the breakdown of cartilage grown in tissue culture (26). Superficially these results are not in accord with those of Ziboh et al (27) who found that aurothiomalate and chloroauine inhzizd the oxygen uwtake of a ohenol-stimulated acetone-pentane powder"of sheep seminal-vesicles by 50"/0 when the enzyme was incubated with arachidonate in the absence of
AUGUST
1975
VOL. 10 NO. 2
245
PRO’STAGLANDINS
glutathione and copper. It appears, therefore, that one or more of these cofactors, which channel the synthesis towards PGE and PGF, might also protect the dioxygenase component of the synthetase complex from inhibition by these compounds. Inconclusion, the results provided by this study show that under the same reaction conditions different anti-inflammatory drugs may either block the synthesis of both PGE2 and PGF2 or selectively inhibit the synthesis of one. If pros? aglandin synthetase in man is similarly affected the difference in response of some patients to either indomethacin or phenylbutazone could be attributable to such different modes of action. The totally unexpected observation that gold salts are completely specific in inhibiting PGF& synthesis whilst stimulating PGE2 synthesis might explain why gold salts are found useful in clinical situations where conventional antiinflammatory drugs are ineffective. Possibly in these situations the "anti-rheumatic" effect of PGE, which reduces lysosomal enzyme release (28) and inhibits experimentally induced polyarthritis in rats (29), may operate. It is tempting to speculate that an important factor which determines the drug of choice for a particular patient is the endogenous ratio of PGE to PGF required for normal function. Patients whose normal function reauires a relatively high PGF level (compared to PGE) would be expected to respond" better to phenylbutazone than to either gold salts or indomethacin and vice versa. However, when considering inflammation, such patients appear to be in a minority and total blockade of all prostaglandin biosynthesis by aspirin or indomethacin is generally beneficial. On the other hand, whilst administered prostaglandin may have a potent effect on blood pressure, inhibition of prostaglandin-biosynthesis by aspirin or indomethacin has little effect. This would suggest that the net effect on blood pressure of opposing acTions of different prostaglandins is insignificant. Thus a drug which acted like the gold salts and increased the ratio of PGE to PGF could be of value in controlling hypertension. REFERENCES 1.
BergstrBm, S., Duner, H., Van Euler, U.S., Pernow, B. & Acta Physiol. Stand. 45, 145-151 Sjovall, J. (1959 > Kaley, G. & Weiner, R. 180, 338-350
(1971)
Crunkhorn, P. & Willis, A.L. 5, 49
Ann. N.Y. Acad. Sci.
(1971)
AUGUST
Brit. J. Pharmacol.
1975 VOL. 10 NO. 2
PROSTAGLANDINS
2.
Rosenthale, M.E., Dervinis, A., Begany, A.J., Lapidos, M. Experientia 26, 1.119-1121 & Gluckman, M.I. (1970) Cuthbert, M.F.
3.
James, G.W.Z. Puglisi, L. p. 219-227,
(1969) (1969)
Brit. Med. J.
",
723
J. Pharm. Pharmac.
2,
379-386
in "Advances in the Biosciences 9” (1973) Pergamon Press, Ed. Bergstrdm
4.
Horton, E.W.
5.
McGiff, J.C. & Nasjletti, A. (;~',~~mi~p~;~sta&andins . and Cyclic AMP", p. 119-151, 9 Kahns & Lands
6.
Crunkhorn, P. and Willis, A.L. Pharmacol. 5, 507-512
7.
Du Charme, D.W. & Weeks, J.R. (1967) in "Nobel Symposium 2",p.173-181, Interscience, Ed. BergstrBm & Samuelsson
8.
Lonigro, A.J., Terragno, N.A., Malik, K.U. & McGiff, J-C. (1973) Prostaglandins 2, 595-606
9.
Giroud, J-P. (1973) Abs. Intern. Meeting on "Future Trends in Inflammation", Verona
10.
Wlodawer, P. & Samuelsson, B. 248 5673-5678 -'
(1973)
J. Biol. Chem.
11.
Wallach, D.P. & Daniels, E.G. Acta 231, 445-457
(1971)
Biochim. Biophys.
12.
Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J. (19
