Current Eye Research
Volume 9 number 7 1990
Hormonal stimulation of 12(R)-HETE, a cytochrome P450 arachidonic acid metabolite in the rabbit cornea Karen L.Davis, Michael W.Dunn and Michal Laniado Schwartzman
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Department of Pharmacology, New York Medical College, Valhalla, NY, USA
ABSTRACT 12(R)-HETE [12(R)-hydroxy-5,8,10,14eicosatetraenoic acid] is one of the major arachidonic acid metabolites produced by microsomal cytochrome P450 of the corneal epithelium. This metabolite is a potent inhibitor of Na+-K+-ATPase activity in several tissues. We investigated endogenous production of 12(R)-HETE in the rabbit corneal epithelium. Incubation corneal epithelial sheets (prelabeled with "C-arachidonic acid) with arginine vasopressin resulted in the production of radioactive 12(R)-HETE suggesting its formation from endogenously labeledarachidonic acid. The maximal response was obtained with 1 pM arginine vasopressin and represents a 15-fold increase in 12(R)-HETE formation compa d with that of control tissues. Stimulation of ''&-arachidonic acid release with a detergent, digitonin, also resulted in endogenous 12(R)-HETE formation. Analysis of the incubation media following digitonin treatment of prelabeled corneal epithelial sheets revealed that 12(R)-HETE production was maximal at 20 pM digitonin, a 17-fold increase over control values. This study is the first to describe hormonal and traumatic stimulation of 12(R)-HETE formation from endogenously labeled arachidonic acid in intact corneal tissues. This study demonstrates that the formation of this Na+-K+-ATPase inhibitor can be modulated by physiological and pathophysiological regulation.
been studied using various preparations of corneal tissues (7).
Cytochrome P450 monooxygenases
comprise an enzyme system consisting of: cytochrome P450 as the hemoprotein; a flavoprotein identified as the NADPH-dependent cytochrome P450 (c) reductase; and phosphatidylcholine which facilitates electron transfer in the microsomal system. Cytochrome P450 is the terminal oxidase existing in multiple forms which differ in substrate, and positional specificity and stereospecificity. Recently, we have reported a novel cytochrome P450 isozyme, P450-AA epoxygenase which selectively metabolizes arachidonate to four regioisomeric epoxides ( 8 ) . In the corneal epithelium, arachidonic acid is metabolized via cytochrome P450 to two biologically active metabolites, 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(R)-HETE) and 12(R)-hydroxy5,8,14-eicosatrienoicacid (12(R)-DiHETE)
(9-11).
12(R)-DiHETE is a potent vasodilator, it disrupts the blood aqueous barrier and has potent angiogenic properties, suggesting that it may have a significant role in promoting corneal inflammation.
INTRODUCTION Three potential pathways for arachidonic acid conversion have been identified: cyclooxygenase,
12(R) -HETE, an endogenous Na+-K+-
ATPase inhibitor, could be a fundamentally important modulator of ocular transport epithelia
lipoxygenase and cytochrome P450 monooxygenases.
that depend on a , ' a N
The specific pathway by which arachidonic acid is
mechanism for their function (9,12).
transformed depends on the tissue, type of stimuli
include: the corneal epithelium and endothelium,
K+-activated ATPase pump Such tissues
and cofactor availability. Several ocular tissues
the ciliary body, and lens subcapsular and retinal
are capable of metabolizing arachidonate via
pigment epithelium.
cyclooxygenase to prostaglandins, thromboxane and
Is 12(R)-HETE an endogenous arachidonic acid
prostacyclin. These include the cornea (1-3),
metabolite? Most of our previous studies were
ciliary body-iris (3,4), anterior uvea (5) and
performed using microsomes isolated from bovine
conjunctiva ( 6 ) .
corneal epithelium to which we added exogenous
The lipoxygenase pathway has
Received on October 27, 1989;accepted on June 28, 1990
@ Oxford University Press
661
Current Eye Research arachidonic acid and cofactors to optimize
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cytochrome P450 activity. This methodology
Arachidonic acid metabolism Rabbit corneal epithelial sheets (111 pg
allowed the characterization of a novel metabolic
of protein; approximately 2 rabbit corneas) were
pathway, isolation of compounds for bioassay and
incubated with 0.4 pCi (7pM) I4C-ara-
identification of their structure. However, it
chidonic acid in 1 ml total volume of PBS
did not address whether endogenous arachidonic
supplemented with NADPH (ImM) at 37OC for 30
acid, esterified into phospholipids in the cell
min.
The reaction was terminated by acidification
membrane, can be oxidized to form 12(R)-HETE under
to pH 4.0 and arachidonic acid metabolites were
basal and /or stimulated conditions. The demon-
extracted with ethyl acetate. Extraction
stration of endogenous formation of 12(R)-HETE in
efficiency was 60-70%. The final extract was
corneal tissues will provide the basis for desig-
dried under nitrogen and resuspended in 200 p l
nating 12(R)-HETE as an endogenous inhibitor of
of methanol. Radioactive metabolites were
Na+-k-ATPase. The aim of this study was to
separated using reverse-phase HPLC.
demonstrate the formation of 12(R)-HETE from
Endonenous arachidonic acid metabolism
endogenously esterified arachidonic acid by hor-
Rabbit corneal epithelial sheets were
monal stimulation or mild trauma, using arginine
preincubated with 7 pM 14C-arachidonic acid
vasopressin (AVP) and a cytotoxic detergent such
for 60 min at 37OC to label cellular lipids.
as digitonin to stimulate arachidonate release.
The nonincorporated 14C-arachidonic acid was removed by washing the tissues with 0.1% bovine
MATERIALS AND METHODS
serum albumin (fatty acid free) in PBS. The
Materials (1-14C)-arachidonic acid
tissue was then placed in PBS supplemented with (56pCi/nunol;
NADPH (1 mM), stimulated with AVP (0.5-4.0 pM)
1Ci-37 GBq) was obtained from Amersham. NADPH and
and incubated at 37OC
arginine vasopressin were obtained from Sigma.
with digitonin (10-40pM) and incubated at
Digitonin was obtained from Aldrich and
37'~ for 10 min.
12(racemic)-HETE from Biomol Research Laboratories
Sevaration of arachidonate metabolites
(Philadelphia, PA).
All solvents were HPLC grade
for 30 min or stimulated
Reverse-phase HPLC was performed on C18
obtained from JT Baker. New Zealand White male
Microsorb column (250 x 4.6 nun) using a linear
rabbits were obtained from Hare Inc.
gradient of 1.25%/min from acetonitrile/water/
PreDaration of fresh corneal eDithelia1 sheets
acetic acid, 50:50:0.1 (v/v/v) to acetonitrile/
Animal use in this study was adherent to the Declaration of Helsinki and The
Guiding
acetic acid, 1OO:O.l (v/v). at a flow rate of 1 ml/min.
Radioactivity was monitored by a flow
Principles in the Care and Use of Animals (DHEW
detector (Radiomatic Instruments & Chemical,
Publications, NIH 80-23). Male New Zealand White
Tampa,FL).
rabbits (1.5-2.5 kg) were anesthetized with
based on previously reported HPLC retention time,
ketamine-HC1 and xylazine.
GC/MS analysis and comigration with a racemic
Topical proparacaine-
HC1 ( 0 . 5 % ) was used to provide local corneal
Identification of metabolites was
mixture of a 12-HETE standard.
anesthesia. Eyes were proptosed and fullthickness sheets of corneal epithelia were surgically
RESULTS
removed using scalpel and forceps. Tissue was
Production of 12(R)-HETE and 12(R)-DiHETE bv
washed and placed in phosphate buffered saline
rabbit corneal epithelial sheets
(PBS), pH 7.4, without calcium. LDH release into
Incubation of corneal epithelial sheets with
the medium was monitored and did not change
I4C-arachidonic acid in the presence of NADPH
significantly during the course of our experiment
resulted in
( 0 - 9 0 min).
metabolites (Fig. 1).
662
the formation of several oxygenated Metabolites formed in
Current Eye Research over 12(R)-DiHETE.
In the rabbit corneal
epithelium 21.5k5.9 ng of arachidonic acid per 111 pg corneal sheets was converted to 12(R)-HETE,
9
whereas only 4.5k2.2 ng of arachidonic acid per
I
n
0
111 pg was converted to the 12(R)-DiHETE.
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2
Metabolite formation was strictly dependent on 6
enzymatic conversion since boiled tissue failed to
2
yield either metabolite.
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a
Endogenous formation of 12(R)-HETE bv rabbit
D
corneal eDithelia1 sheets
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U n
3
We investigated endogenous production of 12(R)-HETE in the rabbit cornea under both physiological (AVP) and pathophysiological J
0
(digitonin) conditions. Rabbit corneal sheets 10
20
30
40
HPLC RETENTION TIME (mid
Figure 1. Reverse-phase HPLC separation of arachidonic acid metabolites formed in rabbit corneal epithelial sheets. Corneal epithelial sheets pg of protein) were incubated with 7 pM [''iifarachidonic acid in the presence of NADPH for 30 min at 37OC. Radiolabled metabolites were extracted and separated by reverse-phase HPLC as described in Methods.
were incubated for 60 min with 14C-arachidonic acid in order to label endogenous lipid pools. Using this procedure 80-85% of the radiolabeled arachidonic acid was incorporated into cellular phospholipids whereas 15-20% was released into the media as metabolites or unmetabolized arachidonate. The prelabeled corneal sheets were then challenged with either AVP or digitonin to release endogenous prelabeled arachidonic acid prior to the formation of arachidonate
rabbit corneal epithelial sheets were identical to
metabolites. Previous studies had indicated that
those previously reported in bovine corneal
maximal conversion of arachidonic acid to
epithelial microsomes and consisted of two polar
12(R)-HETE and 12(R)-DiHETE was achieved in
peaks previously identified as 12(R)-HETE and
corneal preparations at thirty minutes incubation
12(R)-DiHETE.
time. Similarly, AVP-stimulated 12(R)-HETE
This conclusion was based on
comigration with a 12-HETE synthetic standard, and
formation was also time-dependent. The maximal
identical HPLC retention time with 12(R)-HETE and
formation was observed at 30 min whereas shorter
12(R)-DiHETE isolated from bovine corneal
incubations of 10 minutes with AVP also stimulated
epithelial microsomes previously identified by
12(R)-HETE formation but to a lesser extent (data
GC/MS.
not shown).
Metabolites were not formed in boiled
Arginine vasopressin (0.5-4.0 pM)
tissue in the presence or absence of NADPH. Very
maximally stimulated the release of radioactive
low levels of metabolism were seen in intact
12(R)-HETE formation at 1 pM AVP.
corneal epithelial sheets in the absence of NADPH,
12(R)-HETE formation was significantly higher than
an essential cofactor of cytochrome P45O-mediated
control values with all AVP concentrations
reactions (data not shown).
examined (except 0.5 pM).
The formation of
However,
This maximal
12(R)-HETE and 12(R)-DiHETE in the intact
formation represents a 1472% increase in
epithelial sheets was greatly enhanced in the
12(R)-HETE formation compared with unstimulated
presence of NADPH, supporting arachidonate
tissues (Fig 2).
AVP (1 pM) did not have an
metabolism via cytochrome P450 monooxygenases.
effect on the conversion of I4C-arachidonic acid
Under these experimental conditions, arachidonate
to 12(R)-HETE in corneal epithelial microsomes
conversion favored the formation of 12(R)-HETE
(data not shown).
663
Current Eye Research t
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CONTROL
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DIGITONIN (pM)
ARGlNlNE VASOPRESSINCM)
Figure 2. Endogenous formati of 12(R)-HETE following preincubation with “C-arachidonic acid and stimulation by AVP in rabbit corneal epithelial sheets. Cellular lipids corneal epithelial sheets were labeled with “Carachidonic acid for 60 min and subjected to hormonal stimulation with arginine vasopressin in the presence of NADPH as described in Methods. Reaction was terminated and metabolites extracted and analyzed as described. The results are mean & SE; n-4 determinations for 0.5 and 4.0pM AVP and n-5 for 1.0 and 2.0 pM AVP. Statistical analysis by Newman-Keul’s test where p