Sulfasalazine inhibits lyso-PAF: Acetyl-CoA acetyltransferase

Prostaglandins

441245-249,

1992

STJLFASALAZINE INHIBITS LYSO-PAE': ACETYL-COA ACETYTJTRANSFERASE L.D. Faison and H.L. mite Division of Pharmacology, Wellcome Research Laboratories Research Triangle Park, North Carolina 27709 Abstract Sulfasalazine platelet-activating Major metabolites

produced a dose-dependent inhibition of the enzymatic synthesis of factor (PAF) in lysates of rat pleural neutrophils, with an ICsa of 50 PM. of sulfasalazlne, 5-aminosalicylic acid and sulfapyridine, Inhibited this Inhibition of arachidonate synthesis at much higher concentrations.

enzymatic 5-lipoxygenase by sulfasalazine and its major metabolites concentrations (2-3 mM). Because PAF is a potent mediator inhibition of PAF synthesis by sulfasalazine may contribute conditions such as ulcerative colitis and rheumatic illnesses.

was also observed at higher of inflammatory responses, an to its therapeutic actions in

introduction Sulfasalazine is an antiinflammatory agent that has been widely used in the treatment of ulcerative Colitis or inflammatory bowel disease (IBD) for more than 30 years (1). More recently, its mechanism of action has been related to an inhibition of arachidonate metabolism similar to that observed with other salicylates. In particular, both sulfasalazine and its intestinal metabolite, 5-aminosalicylic acid, have been shown to weakly inhibit the 5-lipoxygenase pathway at low millimolar concentrations (2.3). In the present study, we report the effects of sulfasalazine and its major metabolites on lyso-PAF:acetyl-CoA acetyltransferase, the enzyme whrch synthesizes platelet-activating factor (PAF) in response to inflammatory stimuli (4). Materials

and Methods

Pleural

exudate

cells were

harvested

from

male

Sprague-Dawley

(Charles

River)

rats

(150 f 20 g) 3 hr after intrapleural injection of 500 ug carrageenan. Characterization of cell types in these pleural exudates has been described earlier (5). At 3 hr after carrageenan, more than 80% of the cells in exudates were neutrophils. Total white cell counts were obtained from pooled exudates from 5 or 6 rats. Red blood cells were lysed by hypotonic shock as described (5). and washed cells were resuspended at 15-17milliorVml in buffers appropriate for either the acetyltransferase (6) or 5-lipoxygenase (5) assays, as briefly outlined below. Lvso-PAF:acetvl-CoA acetvltransferase. After resuspension in a buffer consisting of 50mM potassium phosphate, 144mM NaCI, 6mM KCI, 5 mM qlucose, 1 mM MqCI,. 1.3 mM CaCl2, pH 7.4, cells were incubated for 5 min at 37OC in the presence of 1;M calcium ionophore A23187 in order to activate the acetyltransferase. Following a centrifugation at 755 g for 2 min at 4°C. drained pellets were resuspended in ice cold glass-distilled water to give approximately 17 million cells/ml and lysed by sonication for 5 set, using a Heat Systems Ultrasonic Processor model W385 with cup horn attachment, power setting 4 (Farmingdale, NY). Portions (0.25 ml) of the resulting lysate were preincubated + potential inhibrtors in 36 mM Tris-Cl (pH 6.9) for 5 min at 37°C before addition of substrates to give 12 UM I~so-PAF, and 2.3 PM [l%]acetyl-CoA (0.06 PCi) in a total volume of 0.50 ml. Blank assays contained non-activated neutrophils and no I~so-PAF. After a 1Omin incubation at 37OC, mixtures were acidified, and [t4C]PAF was extracted using chloroform/methanol as described earlier (6). Aliquots (0.9 ml) of the organic phase were taken to dryness in glass scintillation vials

Copyright

0

1992

Butterworth-Heinemann

246

Prostaglandins

and counted after adding 15 ml EcoLite (ICN Biomedicals, Irvine, CA). Alternatively, chloroform/methanol extracts were dried under nitrogen and chromatographed as described (6) in order to show that [lJC]PAF was the only labeled product extracted when [14C]acetylCoA was used as substrate. Under the conditions employed, product formation was linear with extract concentration and with incubation time for at least 10 min. Arachidonate 5-lipoxvqenase. Cells were resuspended at approximately 15 million/ml in 0.134 M NaCI, 15 mM Tris-Cl, 5 mM glucose, pH 7.4. Aliquots containing approximately 3 million cells were preincubated for 5 min at 37°C in the presence of test compounds, and then, after the addition of ionophore and [14C]arachidonate, for 2 min at 37°C. Product formation was essentially linear during this time (5). Final assay concentrations were 50 mM Tris-Cl, pH 7.4, 1 mM CaCI2, 10 UM [“Ylarachidonate (0.2 PCi), and 3 PM ronophore A23187 in a total volume of 0.40 ml. Products were extracted into ether under acidic conditions and separated by thin-layer chromatography, as described earlier, on Whatman LK5D silica gel G plates,using the solvent system ethyl acetatelisooctanelacetic acid/water (g/5/2/10; top layer after vigorous shaking). This system has been shown to clearly separate the various lipoxygenase and cyclooxygenase products formed from arachidonic acid in neutrophil preparations (5). Leukotriene B4 and 5-HETE, major products of 5-lipoxygenase in this system, were quantitated using scintillation counting techniques. Where inhibition occurred, both of these products were decreased to a similar extent. In some experiments accomplished using a Heat insure complete lysis in the chilling in an ice water bath

cells were lysed before addition of sulfasalazine. This was Systems Ultrasonic Processor as described above. In order to saline-Tris buffer, cells were sonicated for a total of 40sec. with after each 10 set period.

Materials. Sulfasalazine, 5-aminosalicylic acid, sulfapyridine, PAF, and lyso-PAF (L-a-lysophosphatidyl choline, y-0-alkyl) were purchased from Sigma Chem. Co. (St. Louis, MO); calcium ionophore A23187, from Calbiochem Corp. (San Diego, CA); [14C]acetyl-CoA, 56 Ci/mol, from New England Nuclear, Inc. (Boston, MA). [t%]Arachidonic acid, 55 Ci/mol, from Amersham Corp. (Arlington Heights, IL) was purified by silica gel chromatography before use and stored at -20°C under nitrogen (5). Results As shown in Figure 1, sulfasalazine inhibited the enzymatic synthesis of PAF in rat pleural cell lysates with an ICso of 0.05 mM. In this system, PAF synthesis was completely inhibited at concentrations of 0.3 mM and above. The major metabolites of sulfasalazine, Ii-aminosalicylic acid and sulfapyridine, were weak inhibitors of lyso-PAF:acetyl-CoA acetyltransferase, as seen in Figure 1. Extrapolated ICsc concentrations for these metaboliteswere 1.5-2 mM. Inhibition of 5-lipoxygenase by sulfasalazine and its two major metabolites in intact pleural neutrophils was observed at concentrations of 1 mM and above. In these experiments, control values for 5-lipoxygenase activity, measured as DPM ( t 5.E.M.) in the product leukotriene 64, were 19020 + 1310. When cells were lysed by sonication before a preincubation with sulfasalazine, control values were much lower (3876 + 439). However, the inhibitory effect of sulfasalazine was not significantly changed. The 50% inhibitory 5-Aminosalicylic acid and concentration was approximately 2 mM in both cases. sulfapyridine also inhibited 5-lipoxygenase with ICsa values of 2-3 mM.

247

Prostaglandins

10-5

10‘4

10”

MOLAR CONCENTRATION Fiq. 1. Dose-dependent inhibition by sulfasalazine of lyso-PAF:acetyl-CoA acetyltransferase (open circles). Effects of 5aminosalicylic acid and sulfapyridine in the same system are shown as triangles and solid circles, respectively. Data is expressed as mean percent inhibition obtained in 3-6 assays. Control values for PAF = 15800dpm + 490 (S.E.M.). Discussion Synthesis of PAF in various tissues has been shown to occur via two distinct enzymatic pathways (7.8) -- [l] the de nouo pathway, analogous to synthesis of phosphatidylcholine, in which acetylation of an alkyl-phospholipid occurs prior to the sequential removal of phosphate and interaction with CDP-choline and [2] the remodeling pathway in which stimulation of a phospholipase A2 produces lyso-PAF from alkyl choline phosphoglycerides. Lyso-PAF is then acetylated by Iyso-PAF:acetyl-CoA acetyltransferase to form PAF. The arachidonate also released as a product of phospholipase A2 activation may be further metabolized by arachidonate cyclooxygenase and Iipoxygenases. It has been suggested that the de nmm pathway serves to maintain normal endogenous levels of PAF, while the remodeling pathway is thought to be stimulated during proinflammatory and ischemic responses (4). In earlier studies, Stenson and Lobos (2) and Nielson, et al. (3) showed that sulfasalazine and 5-aminosalicylic acid inhibit 5-lipoxygenase in human polymorphonuclear leukocytes at low millimolar concentrations similar to those found to inhibit the enzyme of rat neutrophils in the present study. Their findings supported the use of 5-aminosalicylic acid as a substitute for its parent, sulfasalazine, in the treatment of bowel disease. Others(g) have reported that sulfasalazine and both of its metabolites, at concentrations of 0.5 mM and higher, have radical scavenging effects, which may contribute to anti-inflammatory actions.

248

Prostaglandins

Because earlier

work

cells, it was possible that cell system, inhibitions

similar

to that

of Iipoxygenase

that penetration

relating

study,

inhibition

lipoxygenase

inhibition

was performed might

used for our assays of PAF synthesis. activity were obtained

of cell membranes

In the present

to 5-lipoxygenase

more potent

with either

by sulfasalazine

sulfasalazlne

However,

in a lysed

similar

weak

intact or lysed cells, suggesting

was not a limiting

was found

using intact

be observed

to inhibit

the

factor. remodeling

pathway

of

PAF synthesis in a concentration range quite similar to that reported in human plasma following a single oral dose of 2g (10). Even higher concentrations would probably be attained in the gastrointestinal tract after oral dosing. One might, therefore, expect that inhibition of both PAF and leukotriene synthesis could contribute to actions of sulfasalazrne in the intestine. Our experiments have not shown any effects by sulfasalazine on the ionophore-induced activation of the converts PAF to lyso-PAF (unpublished).

acetyltransferase

or on the

acetylhydrolase

which

It is of particular interest that both PAF and leukotriene 84 have been implicated in inflammatory bowel disease in humans (11,12) and in animal models (13.14). Eliakin and coworkers (12) demonstrated that, in an invitro preparation of colonic mucosa from ulcerative colitis patients, PAF was released during a 4 hr incubation in response to ionophore activation. Both sulfasalazrne and Saminosalicylic acid prevented the release PAF in this in uivo system, with sulfasalazine being at least 2-fold more effective.

of

Our data indicate that sulfasalazine is more potent than its metabolites, as an inhibitor of the enzymatic pathway leading to PAF synthesis in response to inflammatory stimuli. This is consistent with speculations and some clinical evidence supportrng a possible therapeutic advantage for sulfasalazine over its metabolites (15). An inhibition of lyso-PAF:acetyl-CoA acetyltransferase by sulfasalazine may also contribute to its beneficial clinical effects recently reported in psoriaticand rheumatoid arthritis(l6.17). References

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Received: 4-9-92

Accepted: 6-3-92