Synthesis of Slow Reacting Substance-like Activity in
Rabbit Conjunctiva and Anterior Uvea
Prasad 5. Kulkarni and D. Dobli Srinivasan
The ability of rabbit conjunctiva and anterior uvea to synthesize lipoxygenase products was assessed.
Using autoradiographic techniques, we demonstrate that rabbit anterior uvea synthesizes 5 and 12
lipoxygenase products such as 12-HETE, 5-HETE and 5,12-DiHETE and cyclooxygenase product
HHT from 14C-arachidonic acid. Indomethacin pretreated conjunctiva and anterior uvea generated
slow reacting substance (SRS)-like activity from arachidonic acid in the presence of reduced glutathione and A23187. This SRS-like activity contracted guinea pig ileum. Specific SRS-like activity
antagonist FPL-55712 inhibited the contractions of guinea pig ileum induced by SRS-like substance
generated by either conjunctiva or anterior uvea. The activity was still present in the sample following
extraction with organic solvents. SRS-like activity was destroyed by arylsulfatase and its generation
was prevented by either boiling or pretreatment with cyclooxygenase/lipoxygenase inhibitors, BW755
and nordihydroguaiacetic acid. These results indicate that following cyclooxygenase inhibition by
indomethacin rabbit conjunctiva and anterior uvea generate SRS-like activity from arachidonic acid
via lipoxygenase pathways. Invest Ophthalmol Vis Sci 24:1079-1085, 1983
Samuelsson, 1980).8 The presence of the lipoxygenase
pathway, ie, conversion of I4C-AA into 12-HETE, 5HETE, and 5,12-DiHETE (LTB4) has also been demonstrated in the albino rabbit conjunctiva but not in
the iris-ciliary body.12 In this report, we demonstrate
that albino rabbit conjunctiva and anterior uvea synthesize biologically active lipoxygenase products such
as 12-HETE, 5,12-DiHETE, and SRS.
Arachidonic acid (AA) is released from the phospholipid pool of almost all tissues by mechanical or
chemical stimuli1 and quickly converted to cyclic endoperoxides, PGG2 and PGH2 by cyclooxygenase enzyme.2 Cyclic endoperoxides are further converted to
biologically active products such as thromboxane
A2,3 PGE2, PGD2, PGF2,4 and PGI2.5 These cyclooxygenase products are also synthesized in rabbit conjunctiva and anterior uvea from 14C-AA.6 Some cyclooxygenase products such as 6-keto-PGEi, PGE2,
and PGI2 increase intraocular pressure (IOP) and
protein content of the aqueous humor following either topical or intravitreal administration.7
Arachidonic acid is also converted into various
hydroxyeicosatetraenoic acids (HETE) and leukotrienes (LTs) by 12 and 5 lipoxygenase pathways, respectively (see Samuelsson, 1980).8 AA is first converted into an unstable compound 5-HPETE (hydroperoxy eicosatetraeinoic acid) by 5-lipoxygenase,
which is further converted into a group of compounds, the so-called leukotrienes B4, C4, D4, and
E4. Among these agents, LTB4 has been found to be
a potent chemotactic agent for leukocytes.9"1' A mixture of LTC4 and LTD4 represents the slow reacting
substance (SRS) of guinea pig anaphylaxis (see
Materials and Methods
Generation of SRS-like Activity from AA in Rabbit
Conjunctiva and Anterior Uvea
Albino rabbits (1.5-2.5 kg) were killed with Napentobarbital. Conjunctival (approximate wet weights
800 mg) and anterior uveal (approximate wet weight
200 mg) tissues from two animals were excised,
pooled separately, and treated with 10 ng indomethacin for 30 min. Tissues were then chopped and incubated with AA (1 /ug), A 23187 (10 ng), and reduced
glutathione (10 tig) for 1 hr in oxygenated (bubbled
and 95% O2 + 5% CO2) 1 ml Krebs-Henseleit solution
at 37 C. To ensure that cyclooxygenase activity was
completely inhibited, indomethacin (10 jig/ml) was
added to the incubating media.6 Samples were then
centrifuged, and the supernatant tested for its biologic
activity on the bioassay organ guinea pig ileum.
From the Departments of Pharmacology and Ophthalmology,
College of Physicians and Surgeons, New York, New York.
Supported by USPHS Research Grant EY 02861.
Submitted for publication: December 16, 1982.
Reprint requests: Dr. Prasad S. Kulkarni, Columbia University,
630 West 168th Street, New York, NY 10032.
Extraction Procedure
Conjunctival and anterior uveal tissues from three
rabbits were pooled separately and treated with in-
0146-0404/83/0800/1079/$ 1.15 © Association for Research in Vision and Ophthalmology
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1983
Vol. 24
Fig. 1. Generation of SRSlike activity from rabbit
conjunctiva and anterior
uvea following cyclooxy5 cm
genase inhibition: indomethacin (10 /*g/ml) pre,
treated tissues were incubated with arachidonic acid
5 min
and reduced glutathione and
A23187 for 60 min in KrebsHenseleit solution. Tissues
T
T
were separated and the meW
T
T
T
t
w
dia was tested for its biolog30 nq/ml
30 nq/ml
30 nq/ml
30 nq/ml
0.2 ml
ical activity on the guinea
Cch
His
Cch
- His
I.E.
pig ileum bathed in KrebsHenseleit solution containATR -4- DPH
>\
' n ^ ^ MB/ml indomethacin.
u
Figure 1 illustrates contractions induced by carbamylcholine (cch) and histamine (his). In the presence of atropine (ATR) and diphenhydramine (DPH) conjunctival media (CE) and anterior
uveal media (IE) contracted the ileum but cch and his induced contractions were completely inhibited.
domethacin for 1 hr. Tissues were then chopped and
incubated with A A, A 23187 and reduced glutathione
as described above. Samples were mixed with 100%
ethanol. The organic solvent phase was separated by
freezing the aqueous phase on dry ice. The organic
solvent was evaporated and the residue was taken up
in 0.5 ml Krebs-Henseleit solution and tested for its
biological activity.
Bioassay of SRS-like Activity
Guinea pig ileum was used to assay SRS-like activity produced from either conjunctival or anterior
uveal tissues, as described by Stechsehulte et al.13
Guinea pig ileum was suspended in a muscle chamber
containing 5 ml of Krebs-Henseleit solution bubbled
with 95% O2 and 5% CO2. The tissue bathing medium
contained 0.05 jug/ml atropine sulfate, 0.1 ns/m\ diphenhydramine, and indomethacin 1 Mg/ml at 37 C.
Contractions were recorded by an Omniscrib recorder
(Houston Instruments, Texas) using isotonic transducers (Bush Instruments).
Autoradiography
The autoradiography of chromatograms was performed according to the method described by Williams et al.14 The conjunctival and anterior uveal tissues were excised from four animals and pooled separately. Following 30 min of indomethacin (10 ng)
pretreatment tissues were chopped and incubated
with 500 nCi 14C-AA (specific activity 40-60 mCi/
mM) in 1 ml Krebs-Henseleit solution containing 10
jug/ml indomethacin (in order to inhibit cyclooxygenase activity6) for 1 hr at 37 C. Samples were acidified and extracted in chloroform:methanol (2:1). The
organic phase was evaporated and the residue was
reduced under nitrogen to 50 jil and spotted onto thin
layer chromatography (TLC) plates. The chromatogram was then run in ether:hexane:acetic acid
(99:1:0.5) solvent. This solvent system separates AA,
12-HETE, 5-HETE and 5,12-DiHETE. The Rf values
for AA, 12-HETE, HHT and 5,12-DiHETE were 1,
0.78, 0.69, 0.5, and 0.12, respectively, and are identical with the chromatography method used by William et al14 to identify these lipoxygenase products.
The TLC plates were then exposed to x-ray film
(Kodak, X-Omat R film XR-5) for 5 to 10 days. AA,
12-HETE, 5-HETE, 5,12-DiHETE bands were located on the x-ray film and matched on the TLC
plate. The respective bands on the TLC plate were
scraped off, placed in 10 ml of Aquasol scintillation
solvent (New England Nuclear Corp.) and counted
in a Packard scintillation counter.
Results
Synthesis and Bioassay
Figure 1 illustrates that the conjunctival incubation
medium contracted guinea pig ileum in the presence
of atropine (a cholinergic antagonist) and diphenhydramine (a histamine antagonist). Similarly, the
incubation medium of anterior uveal tissue also contracted guinea pig ileum in the presence of cholinergic
and histamine antagonists. Contractions of the ileum
induced by carbachol and histamine were completely
inhibited in the presence of atropine and diphenhydramine, respectively.
Extraction in Ethanol
The guinea pig ileum contracting substance produced by either indomethacin treated conjunctiva or
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No. 8
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LEUKOTRIENE SYNTHESIS IN RABBIT OCULAR TISSUES / Kulkorni ond Srinivosan
Fig. 2. Stability of SRS9
like activity in rabbit conjunctival and anterior uveal
media. Indomethacin pretreated conjunctiva and anterior uvea incubated with
arachidonic acid and re5 cm
duced glutathione and
A23187 for 60 min in Krebs5 min
Henseleit solution and the
T
*
Tw
media was extracted in
0.2 ml
0.4 ml
C
Ac.C.E.
ethanol. Ethanol was evapEtOH
orated under nitrogen and
-EtOH Eitrocts
Extract
residue was reconstituted in
ATR + DPH
1 ml Krebs and assayed on
the guinea pig ileum. Figures b, c, and d illustrate contractions induced by various amounts of anterior uveal ethanol extracts and g represents the contraction of
the tissue induced by 0.2 ml conjunctival ethanol extract. The conjunctival and anterior uveal media were acidified with 1 N HC1 to pH
5 and kept for 1 hr at 75-80 C. The pH was neutralized in 1 N NaOH and assayed for its biological activity. Figure e and h represents the
contractions of the ileum induced by cumulative additions of anterior uveal media and conjunctival media, respectively, after acidification
while Figures a and f represent contractions induced by control by unacidified anterior uveal and conjunctival media, respectively. Figures
I and J represent the conjunctival and anterior uveal tissues were boiled and then incubated with arachidonic acid reduced glutathione and
A23187 for 60 min to Krebs solution failed to generate guinea pig ileum contracting substance.
anterior uvea in the incubation medium can be extracted by organic solvents such as ethanol (n = 3)
and chloroform:methanol (2:1, n = 3). When the extracted substance in ethanol (Figs. 2b-d, g) or in chloroform :methanol (not shown) produced by either
conjunctiva or anterior uvea was tested for its biological activity, it still contracted the guinea pig ileum.
on the guinea pig ileum. Such treatment inhibited the
production of guinea pig ileum contracting substance
(Table 1).
Inactivation of Enzymatic Activity
cyclooxygenase inhibition) produce ileum contract-
Boiling: Two samples of rabbit conjunctival tissue
and two samples of rabbit anterior uveal tissues were
boiled for 1 hr and then incubated with AA, A23187,
and reduced glutathione for 60 min as described
above. When the incubation medium was tested for
its biological activity, it showed no contraction of the
ileum (Figs. 2i, j , Table 1).
Table 1. Characterization of SRS-like activity*
Stability
Arylsulfatase
Acidification: The stability of this substance in acid
media was also tested. The incubation medium of
either conjunctiva or anterior uveal tissues was acidified at pH 5 with 1 N HC1 and kept for 1 hr at 7580 C. The pH of the solution was neutralized by 1
N NaOH and then tested for its biologic activity.
Figures 2a, e, f, and h illustrate that the contractions
induced by the media of conjunctiva or anterior uvea
after acidification were significantly smaller than the
contractions induced by their respective control
media.
Arylsulfatase: Following the incubation period, arylsulfatase (10 and 50 ixg/ml) was added to the incubation medium of either conjunctiva or anterior uveal
tissues for 60 min and then tested for its biologic activity
Lipoxygenase Inhibition by BW755CK and
Nordihydroguaiacetic Acid (NDGA)
Indomethacin (10 n\/m\) treated tissues (following
% Inhibition of SRS-A
synthesis
Treatment
Concentration
Acidification
—
Boiling
50 Mg/ml
100 Mg/ml
BW755
25 Mg/ml
50 Mg/ml
NDGA
10 Mg/ml
50 Mg/ml
FPL 55712
Conjunctiva
Anterior uvea
82 ± 17
n =3
100%
n =4
70 ± 5
n =3
100 ± 0
n =3
24
n = 2
78 ± 13
n =3
46 ± 0.5
n =3
51 ± 1
n =3
90% ± 10
n =3
100%
n =4
68 ± 16
n =3
100 ± 0
n =3
12
n = 2
89 ± 11
n =3
10 ± 2.5
n =3
44 ± 16
n =3
) antagonism of SRS-A like activity
100 ng/ml
67 ± 8
500 ng/ml
1 Mg/ml
* See text.
ND = not determined.
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n =8
N.D.
99 ± 1
n =4
N.D.
47
n
65
n
± 17
=2
± 13
=3
1082
Vol. 24
INVESTIGATIVE OPHTHALMOLOGY b VISUAL SCIENCE / Augusr 1983
/V*
5 cm
-X
iUU
T
0.4 ml
C.E.
C.E.
5 min
s
T
0.4 ml
5cm
T
W
0.4 ml
W
I.E.
5 min
T
0.4 ml
I.E.
50 pg/ml
BW755
50 pg/ml
BW755
T w
0.2 ml
C.E.
ATR + DPH
Fig. 3. Inhibition of the generation of SRS-like activity by
BW755 in rabbit conjunctiva and anterior uvea. BW755 pretreated
(30 min) and control (indomethacin treated) tissues were incubated
with arachidonic acid reduced glutathione and A23187 for 60
minutes in Krebs-Henseleit solution. Each sample was assayed on
the guinea pig ileum. BW755 pretreated tissue samples induced
contractions were significantly smaller than the controls.
ing substance from AA. Therefore, in order to determine that the conjunctiva and anterior uvea (following cyclooxygenase inhibition) synthesize this substance via the lipoxygenase pathway, both types of
tissue were pretreated with (10, 50 Mg/ml) BW755CK
and NDGA, known inhibitors of lipoxygenase and
cyclooxygenase1015 for 30 min prior to incubation.
Figure 3 and Table 1 show that BW755CK at 25 n%l
ml and 50 /xg/ml concentrations inhibited the production of the contracting substance by both tissues
from AA. Similarly, NDGA (10 jig/ml and 50 Mg/ml)
pretreatment for 30 min inhibited the production of
SRS-like activity by both tissues (Fig. 4, Table 1).
5 cm
5 min
T
0.6 ml
C.E.
50 pg/ml
NDGA
50 pg/ml
NDGA
1 pg/ml FPL 55712
ATR + DPH
-H
Fig. 5. Antagonism of SRS-like activity by FPL 55712. Indomethacin pretreated rabbit conjunctiva and anterior uveal tissues
were incubated with arachidonic acid, reduced glutathione, and
A23187 for 60 min in Krebs solution. The conjunctival media
(CE) and anterior uveal media (IE) was bioassayed on guinea pig
ileum in the presence and absence of FPL55712. Thisfigureshows
that the FPL55712 treatment completely inhibited the contractions
of the guinea pig ileum to IE and CE medium.
FPL 55712: Specific SRS-A Antagonist
Figure 5 and Table 1 illustrates that FPL 55712,
a specific SRS receptor antagonist,16 antagonized the
contraction of guinea pig ileum induced by a substance produced by either conjunctiva or anterior
uvea in a dose-dependent fashion.
Lipoxygenase Product Formation from 14C-AA by
Rabbit Anterior Uvea
Figure 6 (radiochromatogram) and Table 2 show
that rabbit anterior uvea synthesized 12-HETE, 5HETE and 5,12-DiHETE (lipoxygenase products)
and HHT (a cyclooxygenase product) from 14C-AA
(500 nCi). Although other lipoxygenase products, ie,
5,12-DiHETE and 5-HETE synthesized by the rabbit
anterior uvea and conjunctiva were in equal amounts,
the 12-lipoxygenase product, 12-HETE, synthesized
in the conjunctivas was three- to four-fold more than
in the anterior uvea (Table 2).
Discussion
ATR + DPH
Fig. 4. Inhibition of the generation of SRS-like activity by nordihydroguaiacetic acid (NDGA) in rabbit conjunctiva and anterior
uvea. Both experimental tissues were pretreated with NDGA and
indo while control tissues were pretreated with indomethacin alone.
Both tissues were incubated with arachidonic acid, reduced glutathione, and A23187 for 60 min in Krebs-Henseleit solution and
each sample was assayed on guinea pig ileum. Contractions of the
ileum induced by NDGA pretreated tissue samples are significantly
smaller than its respective controls.
0.2 ml
I.E.
Our results clearly show that rabbit anterior uvea
has the ability to synthesize lipoxygenase products in
contrast to previous results12 that indicated that rabbit
iris-ciliary body had no ability to synthesize lipoxygenase products. The different results may be explained by differences in experimental technique. For
example, in previous studies12 only one anterior uveal
tissue was incubated with 14C-AA for 30 min. In the
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LEUKOTRIENE 5YNTHESI5 IN RABBIT OCULAR TISSUES / Kulkorni and Srinivasan
No, 8
present study, we pooled four anterior uveal tissues
and incubated the tissues with I4C-AA for 60 min.
In addition, radioactive peaks of the various product
on the TLC plates were located using radioscan in
the previous study12 whereas we exposed the TLC
plate to x-ray film for 10 days. Since the total amount
of lipoxygenase product formed in the iris-ciliary
body is about half as much as that formed in conjunctiva, it is possible that radioscanning technique
could have detected products found in only conjunctiva but not in the iris-ciliary body of rabbits.
Although we demonstrated the presence of a lipoxygenase pathway in both conjunctiva and anterior
uvea, cyclooxygenase activity in the anterior uvea
appears to be more potent than lipoxygenase activity
because only 7-8% lipoxygenase products were formed
from 14C-AA whereas cyclooxygenase product formation was about 12-20%.6 Both tissues produce 12HETE, 5,12-DiHETE, and 5-HETE indicating the
presence of both the 12- and 5-lipoxygenase activities.
In a previous study, we showed that steroidal antiinflammatory drugs such as dexamethasone and betamethasone inhibited many signs of intraocular inflammation including leukocyte infiltration into the
anterior chamber induced by bovine serum albumin
(BSA).17 However, paradoxically, topical indomethacin (0.5%) treatment potentiated the leukocytic response while it reduced conjunctival and iris hyperemia and aqueous flare.17 We further demonstrated
that 0.5% indomethacin only partially inhibited cyclooxygenase activity of the rabbit anterior uvea.18
Thus, it is possible that following partial or complete
cyclooxygenase inhibition the synthesis of lipoxygenase products is facilitated. Since some of these products such as 5,12-DiHETE (5-lipoxygenase product),
which can be synthesized by rabbit anterior uvea, are
now known to be markedly chemotactic,9"19 it is
not surprising that indomethacin (0.5%) potentiated
the leukocyte response in the BSA model.
The name slow-reacting substance (SRS) was introduced by Feldberg and Kellaway20 for a smooth
muscle contracting factor in the perfusate of cat and
guinea pig lungs following treatment with cobra
venom. Subsequently, several investigators demonstrated the release of SRS from different tissues, such
<-AA
^-12-HETE
<-5-HETE
<-5,12-DIHETE
LIPOXYGENASE PRODUCTS IN
RABBIT ANTERIOR UVEA
Fig. 6. Autoradiograph of lipoxygenase products synthesized
from l4C-arachidonic acid by rabbit anterior uvea. This figure
shows that rabbit anterior uvea synthesized different lipoxygenase
and cyclooxygenase products from uC-arachidonic acid. Rf values
for AA, 12-HETE, HHT, 5-HETE, and 5,12-DiHETE are 1, 0.78,
0.69, 0.5, and 0.12, respectively.
as platelets,21 lung,22"25 heart,26 rat basophilic leukemia cells (RBL-1),27 and leukocytes28"30 of different
species.
Table 2. % Lipoxygenase product formation in rabbit ocular tissues from
14
C-Arachidonic acid*
Tissue
5,12-DiHETE
5-HETE
HHT
12-HETE
Total
Anterior uvea
n=3
Conjunctiva
n=3
2.43 ± 0.08
2.8 ± I.I
1.2 ± 0.1
2.1 ± 0.6
7.3 ± 0 . 5
1.7 ± 0.6
2.0 ± 0.7
8.0 ± 3.5
11.5 ± 3.7
. 2.0 ±0.6
* Each incubation carried out at 37 C for 60 min in 1 ml of Krebs-Henseleit
solution and amount of AA used was 500 nCi (sp. activity = 40-60 mCi/
mM). Numbers in the columns represent % of the total extracted radioactivity
counts.
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / Augusr 1983
The characterization of the release of SRS-like activity by several investigators was determined as follows: (1) SRS-like activity is smooth muscle contracting (ie, guinea pig ileum) activity20; (2) arachidonic
acid is the precursor and is converted into SRS via
the lipoxygenase pathway27-31; (3) SRS-like activity
can be extracted in organic solvents21'32'33; (4) SRSlike activity is unstable at acidic (pH < 5) medium31;
(5) arylsulfatase, a proteolytic enzyme, destroys
15
SRS31,33,34. (6) nordihydroguaiacetic acid (NDGA)
and BW755, the cyclooxygenase/lipoxygenase inhibitors10 should reduce the production of SRS-like activity and boiling should denature and inactivate the
enzymatic activity to produce SRS; and (7) FPL
55712, a specific antagonist of SRS16>26>35 antagonizes
the smooth muscle contracting substance induced
by SRS.
Our results clearly indicate that following cyclooxygenase inhibition the rabbit conjunctiva and anterior uvea have the ability to synthesize a guinea pig
ileum contracting substance with SRS-like activity
from arachidonic acid in the presence of co-factors
A23187 and reduced glutathione.
SRS is a mixture of leukotriene C4 and D4. Both
of these compounds are thought to be mediators of
allergic and anaphylactic reactions.36"38 They also
cause vasoconstriction39 and cardiac arrest.26 At present, however, the ocular effects of these compounds
are not known.
Key words: SRS-like activity, lipoxygenase, cyclooxygenase,
12-HETE, rabbit conjunctiva, anterior uvea
Acknowledgments
The authors wish to thank Ms. Ana Rodriguez, Ms.
Helen Kahng, and Mrs. Carolyn Rudeck for their technical
assistance and Ms. Ann Zaragoza for typing this manuscript.
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