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Effect of Gold Salts and Other Drugs on the Release and Activity of Lysosomal Hydrolases.

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Effect of Gold Salts and Other Drugs on the
Release and Activity of Lysosomal Hydrolases
By ROBERT
S. ENNIS,JOSE LUISGRANDA,
AND AARON
S. POSNER
Acetylsalicylic acid, indomethacin, phenylbutazone, gold thiomalate and gold
thioglucose had no protective effect on
the release of hydrolases from lysosomes.
Only the gold compounds produced a
marked inhibition of acid phosphatase,
8-glucuronidase and cathepsin, obtained
from both rabbit liver lysosomes and human synovial fluid. This inhibition is reversible by the addition of a sulfhydryl
compound. It is suggested that a possible mechanism of gold action in
rheumatoid arthritis is the inhibition of
intra-articularhydrolases.
I
T has been postulated that the final stage
in the development of rheumatoid joint
disease is the disruption of intra-articular
lysosomes, with subsequent release of free
lysosomal hydrolases, followed by degradation of the joint cartilage.1.2.3Evidence supporting this concept has been obtained from
both morphological and biochemical investigations. Histochemical methods h a w
shown lysosomes in joints to be present in
a ) the lining cells of the synovial membrane: b ) in wandering and fixed tissue
macrophages,5 and c) in polymorphonuclear leukocyte^.^.^ Biochemical studies have
shown increased titers of lysosomal acid hydrolases in rheumatoid synovial fluid. In
particular, acid phosphatase,8 p-glucuronid a ~ eand
, ~ P-acetylglucosaminaselO have all
been identified in these pathological joint
fluids.
Z 8 et al.ll have shown the presence of a
neutral protease in extracts of human polymorphonuclear leukocytes and rheumatoid
synovial membrane capable of hydrolysing
proteinpolysaccharides. In addition, Ali et
al.12,13
and Fell and Dingle14 have shown
that an acid protease, tentatively identified
as cathepsin, is capable of degrading the extracellular matrix of joint cartilage. In addition, substances which cause the release
of lysosomal enzymes, such as streptolysin
S,'; and endotoxin,lGwhen injected directly
into joints of experimental animals, produce
a chronic arthritis characterized by synovial
hyperplasia, focal concentrations of lymphocytes, and pannus formation. Thus, it has
been proposed that various anti-inflammatory drugs exert their therapeutic effects
by stabilizing lysosomes, thereby retarding
the liberation of hydrolytic enzymes into
the synovial fluid.3 In the case of gold salts,
Persellin and Z s 5 suggested from studies
on peritoneal macrophages, that direct inhibition of hydrolytic enzymes is a possible
mechanism of their anti-inflammatory action.
Publication Number 52 from The Laboratory of
Ultrastructural Biochemistry, Hospital for Special Surgery, Cornell Unicersity Medical College,
New York, N . Y. 10021.
This work supported in part by PHS Grant DE01945 from the National Institute of Dental Research.
ROBERTS . ESSIS, M.D.: Resident in Surgery,
Neu; York Hospital. Cornell University Medical
College. JOSE LUIS GRANDA,M.D., PH.D.: Assistant Professor, Department of Surgey, Cornell
University Medical College. AARON S . POSNER,
PH.D.: Associate Director of Research, Hospital
for Special Surgery, Associate Professor of Biochemistry, Cornell University Medical College.
Address reprint requests to Dr. Posner.
756
ARTHRITIS AND RHEUMATISM, VOL.
11, NO. 6 (DECEMBER 1968)
75;
DRUGS AND LYSOSOMAL HYDROLASES
80
-
lL
i'
E ao-
lu
c
4.0
-
20-
2
3
4
5
PH
Fig. 1-The pH dependance of the proteolytic activity of human rheumatoid
synovial fluid. Enzyme activity in arbitrary units.
It was the object of this work to determine whether proteolytic activity is present
in the synovial fluid of patients with rheumatoid arthritis, and to study the effect of
gold salts and other drugs used in the treatment of rheumatoid arthritis on the release
and activity of lysosomal hydrolases. A preliminary report of these findings has appeared.17
MATERIALSAND METHODS
Synovial fluid was aspirated from the knee joints
of patients with rheumatoid arthritis and immediately cooled to 0 C. Samples grossly contaminated with blood were discarded. The fluid was
centrifiiged withoiit delay at 2000 rpm. for 5 min-
iites at 0 C. to remove cellular components. Enzyme assays were then performed in the manner
described below.
Intact lysosomes were separated from the fresh
rabbit liver tissue by differential ultracentrifugation at 0" C. in 0.25M sucrose, according to the
method of De Duve et al.18 The lysosomd stability of this fraction in the presence of a number
of test substances, was measured by the technique
of De Duve et al.19 One part by volume of the
test substance dissolved in 0.25M sucrose was
added to a solution containing 10 volumes of the
liver particle suspension, and 10 volumes of 0.1M
acetate buffer (pH = 5.0). When a non-aqueous
solvent was used, a separate aliquot of particle
suspension was incubated with solvent alone to
serve as a control. The test mixtures were preincubated for 30 minutes at 37 C.after which en-
758
ENNIS, GRADA, POSNER
0Synovial Fluid t Cystrinr
Synovial Fluid t W d Thiomalak
Spwial Fluidt Qold Thiamolotrt Cystrinr
ACID PHOSPMATASE
CATMEPSIN
1
Fig. 2-Effect
of gold thiomalate (1.0 mg./ml.) and of cysteine (10 mM/L)
on the activities of synovial fluid acid phosphatase a n d cathepsin.
zyme substrate prepared in 0.25M sucrose and
0.1M acetate buffer was added. Acid phosphatase
activity was used as a measure of the degree of
lysosomal disruption a t p H 5.0. The following test
substances were used in the above procedure:
1.) Hydrocortisone sodium succinate (SolucortefB, The Upjohn Co.) in concentrations ranging
from 5.0 mg./ml. to 0.1 mg./ml.
solution in 95
per cent ethanol.
2.) Acetysalicylic acid in concentrations ranging
from lO-3M to lO-GM, either as aqueous suspension or in solution of 50 per cent ethanol.
3.) Indoniethacin (Indocin@, Merck, Sharp, and
Dohme Inc.) in aqueous solution was tested in
concentrations ranging from 2 mg./ml. to 0.02
mg./ml.
4.) Phenylbutazone (ButazolidinB, Ceigy) was
tested in aqueous suspension of 5.0 mg./ml.
5.) Gold Sodium Thiomalate (MyochrysineB,
Merck, Sharp, and Dohme) and Gold Sodium
Thioglucose (SolganalB) were tested in concentrations ranging from 5.0 mg./ml. to 0.01 mg./nil.
The same analyses for enzyme content and inhibition were employed for both synovial and
lysosomal solutions. Acid phosphatase was assayed
by the method of Andersch and Szezypinski,20
f3-glucuronidase and cathepsin were measured according to the method of Gianetto and D e Duve.21
The cathepsin unit is defined as that quantity of enzyme which in 60 minutes produces a
colored substance not precipitable by trichloracetic
acid, equivalent to 1 milli-equivalent of trymine.
The acid phosphatase and f3-glucuronidase units
are defined as the mM of the product liberated
per 60 minutes’ incubation.
The pH dependance of the synovail fluid protease was tested with the following buffers: HCLKCL, pH 2.0; Glycine-HCL, p H 2.4 and p H 2.8;
acetic acid-sodium acetate, pH’s: 3.6, 4.0 and 5.0.
The total enzyme activity of lysosomal preparations was determined using the above assay procedures after addition of 0.1 per cent Triton
X-100 detergent to disrupt intact particles. To
assay enzyme inhibition, a series of gold thio-
759
DRUGS AND LYSOSOMAL HYDROLASES
Table 1.-Relative Concentrations of Acid Phosphatase and Cathepsin
Denionstrated in Human Knee Pathological Synooial Fluid
Volume
in ml.
Patient
5.2
B.I.
2.5
R.G.
3.4
1l.R.
6.2
L.K.
4.1
M.W.
4.2
J1.A.
4.0
E.G.
Diagnosis
Osteoarthritis
(with effusion)
Posetraumatic
arthritis
Rheumatoid
arthritis
Rheumatoid
arthritis
Rheumatoid
arthritis
Rheumatoid
arthritis
Rheumatoid
arthritis
Cathepsin
units */ml. $
Acid phosphatasc
unitst/ml.
1.07
1.19
2.38
0.96
2.42
-
2.34
1.49
3.72
2.20
2.86
2.40
3.4
2.33
*Cathepin unit =milliequivalents of tryosine std. released in 60 minutes a t p H 3.6 and 37" C.
tAcid phosphatase unit =mM of p-nitrophenol released in 60 minutes p H 5.0 and 37" C.
$ml. of synovial fluid.
malate solutions (ranging from 0.01 to 5.0 mg./ml.)
were incubated with enzyme solutions (30minutes
pH 5.0, 37 C) prior to the substrate addition.
Finally, to test the reversibility of the gold inhibition, cysteine a t a final concentration of 10 mM,
was added to the enzyme system which had been
preincubated with gold thiomalate.
In an effort to establish the mechanism of this
inhibition, detailed kinetic studies (using Lineweaver and Burk plots) were performed on acid
phosphatase and P-glucuronidase liberated from
rabbit liver lysosonies after treatment with double
distilled, deionized water. The following inhibitors
were added to the enzyme system in each of the
experiments: gold thiomalate, 2.5 mM, gold thioglucose, 2.5 mM, iodoacetate, 5.0 mM, mercuric
acetate, 5.0 mM, pchloro-mercuric benzoate, 5.0
mM and sodium fluoride, 5.0 mM. After adding
the inhibitor the enzyme solutions were preincuhated for 30 minutes a t 37 C, and pII 5.0. The
solutions containing iodoacetate and p-chloro-mercriric benzoate, on the other hand, were preincubated in glycine buffer at pH 9.0 for 33 minutes
at 37 C: and prior to the addition of substrate the
solution p H was returned to 5.0 by the addition
of 0.2M acetic acid.
RESULTS
Stynooiul Fluid Hydrolases
A cathepsin-like acid protease can be
demonstrated in human rheumatoid synovia1 fluid. The pH dependence of this protease is shown in Fig. 1. It can be seen that
it has a pH optimum of 2.5-2.8. The enzyme is slightly activated by the addition
of lOmM cysteine (Fig. 2). The relative
concentrations of cathepsin and acid phosphatase, obtained from pathological synovial fluids, are shown in Table 1.
E f e c t of Anti-injlammatory Drugs on the
Release and Activity of Lysosomal Hydrolases
The effects of hydrocortisone, acetylsalicylic acid, indomethacin, phenylbutazone
and gold salts on the release of acid phosphatase from rabbit liver lysosomes are
summarized in Table 2. Actually, when gold
thiomalate, in aqueous solution, was incubated with intact lysosomal particles in concentrations ranging from 5.0 mg./ml. to
0.01 mg./ml., the free enzyme activity was
found to be markedly diminished. This apparent protective effect of gold on the lysosoma1 membrane, however, could be ac-
760
ENNIS, GRADA, POSNER
Table t.--Eaect of Anti-inflammatory Drugs on the Release
Acid Phosphatase from Rabbit Liver Lysosomes
Drug
Control
Hydrocortisone
Acetylsalicylic acid
Indomethacin
Phenylbutazone
Gold thiomalate
of
No. exp.
Free activity
(% of total)
13
6
6
8
4
14
35.5 f4.7
27.5 k 3.5
40.0 f4.2
45.8 f5.3
85.1 f5.0
43.0 *4.6*
*Corrected for direct enzyme inhibition as shown in Table 3. All other drugs showed no inhibition.
? =Standard deviation.
counted for entirely by a direct inhibitory
effect upon the enzymes. All other drugs in
Table 2 showed no free enzyme inhibition
and thus did not require t h i s correction.
The experiments reported in Table 2 represent the average of the three different drug
concentrations outlined above in Materials
and Methods. All of the results, for a given
drug, were averaged, since no significant
difference in free activity of acid phosphatase was found at the different drug concentrations. Thus, it can be seen that only
hydrocortisone produces any significant
protective effect, while phenylbutazone
alone has a marked disruptive effect, on the
lysosomal membrane.
Inhibition of Hydrolases by Gold Salts
The relative effectiveness of gold thiomalate on the inhibition of synovial fluid and
rabbit liver lysosomal acid hydrolases is
shown in Table 3. Gold thioglucose and
gold chloride were found to be identical
to gold thiomalate in the inhibition of lysosoma1 hydrolases in the range of concentrations tested. The effect of the concentration of gold in the inhibition of acid
phosphatase and p-glucuronidase is shown
in Fig. 3. It can be seen that at concentrations as low as 0.01 mg./ml. there is still
s i w c a n t inhibition of P-glucuronidase.
Mechanism of Gold Inhibition
Enzyme inhibition by the gold compounds was found to be independent of pH
or precipitation effects. In addition, the
chelating agent, EDTA (10 mM/L) had
no effect on the activity of the lysosomal
hydrolases, suggesting that the gold is not
competing with other metals required for
enzyme activity.
The sulfhydryl-binding agents, mercuric
acetate, p-chloro-mercury benzoate and iodoacetate produced a similar inhibition
when incubated with the free enzymes.
Acid phosphatase was reduced in activity
Table 3.-Enzyme Inhibition b y Gold Thiomalate, 1.0 mg./ml., at
pH 5.0,37C, Pre-incubated for 30 Minutes
Enzyme
Acid phosphatase
p-glucuronidase
Cathepsin
Percent inhibition
Rabbit liver lysosomes
64.5%f3.9% (7)*
32.5%+5.2% (5)
30.0%k4.3% (4)
*Number of experiments shown in parentheses.
+Standard deviation.
H~~~~
synovial fluid
48.4%f6.1% (5)
50.2%f3.1% (2)
62.5%f4.5% (5)
761
DRUGS AND LYSOSOMAL HYDROLASES
Pre-Incubation, pH 4.8, 37OC, 4 0 min.
Acid Phosphotose
p-Glucuronidose
Control
0
5.0 mg Gold
g Liver
2.5 mg Gold
g Liver
1.0mg Gold
g Liver
0.5 mg Gold
0.1mg Gold
g Liver
1 Li
rl
g Liver
1
inhibition o
I.-Effect of gold concentration in thc
acid phospharase and
fl-glucuronidase of rabbit liver lysosomes. The enzymes from a gram of liver tissue
are dissolved in 10 ml. of buffer for each gold concentration.
by 82.5 per cent, 57.0 per cent and 45.0
per cent respectively, by these agents. In
like manner, it was shown that the addition
of a sulf-hydryl compound (cysteine, 10
mM/L) to the system, following preincubation with gold thiomalate, produced reversal of the enzyme inhibition (Fig. 2 ) .
Acid phosphatase assayed at pH 5.0, 37
C and 10 minute incubation with p-nitrophenylphosphate yielded a K,,, of 8.48 x
10-4M. The K, for P-glucuronidase, incubated for 30 minutes at pH 5.0, 37 C, was
found to be 1.76 X 10-3M. A plot of S-l
versus V-l (Fig. 4) demonstrates that iodoacetate, mercuric acetate, gold thioglucose and sodium fluoride all act as noncompetitive inhibitors of acid phosphatase.
The action of sodium fluoride, a known noncompetitive inhibitor of acid phosphatase, is
not reversed by the addition of sulfhydryl
compounds. Finally, Table 4 shows that
gold compounds are weak inhibitors of acid
phosphatase when compared to mercuric
acetate, iodoacetate and sodium fluoride.
DISCWSSION
These investigations show that a proteolytic enzyme, which has been identified in
human rheumatoid synovial tissue,22 is also
present in significant amounts in rheumatoid synovial fluid. This enzyme has a different pH optimum from the one described
by ZifE et a1.I1 in polymorphonuclears and
rheumatoid synovial membrane and it is
probably a different enzyme. The difference between the pH optimum (2.5-2.8)
of this rheumatoid synovial fluid protease
'and the pH optimum (4.0) of a similar
protease found in polymorphon~clears,~~
is
probably due to the different substrates
employed. The pH dependance of the enzyme was not measured above a pH of 5.0
762
ES-NIS, GRADA, POSNER
9
30
-
20
-
10
I
0-
-n
-10
-6
-
2
0
2
6
n
~
I
I
I
I6
al
26
s" I d
and Burk Plot of inhibitors of acid phosphatase from rabbit
liver lysosomes. Pre-incubation 30 minutes, followed by 10 minute incubation at
pH 5.0,37" C, using p-nitrophenylphosphatesubstrate.
Fig. 4-Lineweaver
because of technical difficulties. When heatdenatured albumin is used as a substrate
above a pH of 5.0, the filtration time of the
TCA precipitate is so long (24 hours) that
acid hydrolysis of the protein occurs. Finally, the proteolytic activity found in the synovial fluid is tentatively identified as cathepsin D on the basis of pH optimum and
slight activation by sulphydryl groups.
There were no differences in the levels
of cathepsin in the synovial fluid of patients
with rheumatoid and post-traumatic arthriConstants, Ki,of Certain
Compounds on Acid Phosphatase from
Rabbit Liver Lysosomes
Table 4.-Inhibidon
Ki
ComDound
Gold thiomalate 2.5 mM
Gold thioglucose 2.5 mM
Mercuric acetate 5.0 mM
Iodoacetate 5.0 mM
Sodium Fluoride 5.0 mM
1.39 X
3.15 X
1.82 x
1.69 x
6.92
x
1O-zM
10-2M
10-5~
10-5~
10-5~
tis, and even in the case of a patient with
osteoarthritis, who had an inflammatory
component, there was considerable cathep
tic activity in the synovial fluid. These results stress the point that the liberation of
hydrolytic enzymes is a consequence of the
inflammatory process regardless of the origin. The levels of cathepsin found in the
pathological synovial fluids suggest strongly
that this enzyme may play a role in cartilage destruction. Concomitant acid phosphatase measurements are in agreement
with levels previously reported in the literatureSs
Of the drugs tested, only hydrocortisone
was capable of producing a significant protective effect upon intact lysosomes subjected to artificial stress conditions. These
findings with hydrocortisone are in agreement with the in uitro studies of De Duve,lB
and the in vivo work of Weissmann and
763
DRUGS A N D LYSOSOMAL HYDROLASES
Th0mas.2~On the other hand, acetylsalicylic acid was found to have no protective
effect upon isolated lysosomes at pH 5.0,
in agreement with the report of Weissmann.25 However, a previous report indicates that enzyme release may be retarded
by this drug, presumably, at a higher pH
value.26The activation of lysosomes by phenylbutazone is difficult to interpret since
the drug was incubated with the lysosomes
as a suspension and not in solution. Another
example of the disruption of lysosomal
membranes by suspended particles has
been given in an earlier study of the cytotoxic effects of silica on macrophages.27 Finally, it should be stressed that the experimental results suggest that anti-inflammatory drugs effective in treating rheumatoid
arthritis do not necessarily act through direct stabilization of lysosomal membrane.
The use of gold oompounds in the treatment of rheumatoid arthritis has been
thoroughly reviewed by Freyberg.28 Investigations by Persellin et al.29have shown
that gold salts do not alter or retard the
immune response in rabbits challenged by
bacterial products. Early studies indicated
that gold salts have a definite chemotherapeutic effect against hemolytic streptococci,
tubercle bacilli and other bacteria.28 The
ability of gold salts to prevent PPLO-induced chronic arthritis in mice, has also
been known for some time, but the same
gold compounds have no effect on the in
vitro growth of these organisms.2BThese
observations have led some investigators28
to postulate that the gold salts may act
non-specificallyby altering the enzyme systems of host cells.
Persellin and Zig5 have shown that gold
is taken up by tissue macrophages, and
that it is accumulated in lysosomes. They
found in addition that the gold salts inhibited the activities of acid phosphatase and
P-glucuronidase. The present experiments
show quantitatively that gold compounds
are potent inhibitors of acid phosphatase,
/3-glucuronidase and cathepsin. I n vitro inhibition has been demonstrated in the case
of /3-glucuronidase (Fig. 3) at gold concentrations comparable to those found in
the plasma during gold therapy.2Y
From these experiments, the mechanism
of gold inhibition is presumed to be via
the binding of sulfhydryl groups. This cunclusion is based on a ) the similarity in a c
tion of gold salts to enzyme inhibitors
known to act by sulfhydryl binding, and
b ) the reversibility of this inhibition by the
addition of cysteine. In this light, Libensonv2reported that the toxicity of a series
of gold compounds could be correlated with
their relative sulfhydryl binding capacities.
Additionally, he showed that reduced glutathione produced a decreased toxicity of
gold salts in the rat. The lack of activation
of acid phosphatase or p-glucuronidase, and
the minimal activation of cathepsin D by
sulfhydryl compounds suggest that this inhibition is due to a non-specific alteration
of enzyme protein structure.
SUMMARIOIN INTERLINGUA
Esseva constatate que acido acetylsalicylic, indomethacina, phenylbutazona, thiomalato de auro, e thioglucosa de auro exerce nulle efFecto protective super le liberation de hydrolases ab lysosomas. Inter le agentes mentionate, solo le compositos a auro
effectuava un inhibition marcate de phosphatase acide, p-glucuronidase, e cathepsina
obtenite ab lysosomas hepatic de conilio e ab human fluido synovial. Iste inhibition
es reversibile per le addition de un composito sulphhydrylic. Es suggestionate que le
inhibition de hydrolase intra-articular es un mechanism0 possibile subjacente le effect0
de auro in arthritis rheumatoide.
REFERENCES
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764
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ENNIS, GRADA, POSNER
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27. Allison, A. C., Harington, J. S., and Birbeck,
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of gold salts. Exp. Med. Surg. 3:146, 1945.
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