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)


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


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


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


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


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


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


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).


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,


whereas only 4.5k2.2 ng of arachidonic acid per




111 pg was converted to the 12(R)-DiHETE.


E a U


Metabolite formation was strictly dependent on 6

enzymatic conversion since boiled tissue failed to


yield either metabolite.



Endogenous formation of 12(R)-HETE bv rabbit


corneal eDithelia1 sheets


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U n


We investigated endogenous production of 12(R)-HETE in the rabbit cornea under both physiological (AVP) and pathophysiological J


(digitonin) conditions. Rabbit corneal sheets 10





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


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


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


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).


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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

Hormonal stimulation of 12(R)-HETE, a cytochrome P450 arachidonic acid metabolite in the rabbit cornea.

12(R)-HETE [12(R)-hydroxy-5, 8, 10, 14 eicosatetraenoic acid] is one of the major arachidonic acid metabolites produced by microsomal cytochrome P450 ...
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