Exp. Eye Res. (1992)
55, 235-241
Metabolism
of Arachidonic
KARA L. KING”,
Acid Epithelium
by Isolated
NICHOLAS A. DELAMERET, STEPHEN AND WILLIAM M. PIERCE, JR.”
Rabbit
Ciliary
C.CSUKAS
Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Research Institute and aDepartment of Pharmacology, and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A. (Received Chicago 22 March 1991 and accepted in revised form 21 November 7991) We examined the ability of rabbit ciliary epithelium to metabolize arachidonic acid in vitro. The epithelium was homogenizedand incubatedwith l*Clabeled arachidonicacid. **C-labeledmetabolites were extracted and then separatedby thin layer chromatography. The range of arachidonic acid metabolitessynthesizedby ciliary epitheliumwas comparedto the metabolitesgeneratedby rabbit irisciliary body. Ciliary epitheliumproducedsubstantialamountsof arachidonic acid metabolitesthat comigrated with 5-HRTFand 12-HRTE.Authenticity of the 12-HRTEproducedby ciliary epithelium was confirmed by gas chromatography/massspectrometry. The ciliary epithelium generatedonly small amountsof the cyclooxygenaseproducts,PGF,,, PGE,,PGD, and Gk-PGP,,.In contrast, the iris-ciliary body producedlarge amountsof cyclooxygenaseproducts such as PGF,, and PGD,. The ability of the ciliary epithelium to generate 12-HETEis noteworthy since 12(R)-HRTFis known to be capableof lowering intraocular pressure. Key words:ciliary epithelium; iris-ciliary body: rabbit; arachidonicacid: HETES;prostaglandins.
1. Introduction Arachidonic acid can be metabolized by three pathways : cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase. The metabolites of arachidonic acid can vary from one tissue to another, probably due to the different prominence of each specific metabolic pathway within diierent cell types. For example, the cornea and iris both appear capable of metabolizing a substantial amount of arachidonic acid by the lipoxygenase pathway (Kulkarni, Fleisher and Srinivasan, 1984; Hurst et al., 1989) while lipoxygenase activity in the lens is much lower (see Bazan, 1989). In fact, Williams, Delamere and Paterson (1985) failed to detect the generation of lipoxygenase products from exogenous arachidonic acid by either rabbit or frog lens. Arachidonic acid metabolites are well known for their role in inflammatory
responses of the eye
(Bhattacherjee, 1989). Recently, one particular arachidonic acid product, 12(R)-hydroxyeicosatetraenoic acid [ 12(R)-HETE] has received attention because it can lower intraocular pressure (Masferrer, Dunn and Schwartzman, 1990). Since 12(R)-HETE can inhibit Na,K-ATPase activity in cornea and other tissues (Schwartzman et al., 198 7 ; Escalante et al., 1988) it has been suggested that the * Present address : Discovery Research, Allergan Pharmaceuticals 2525 Dupont Drive, Irvine, CA 92715, U.S.A. t For reprint requests and correspondence at: Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville. KY 40292, U.S.A.
00144835/92/080235+07
%08.00/O
TOP-lowering effect of 12(R)-HETE might be related to the inhibition of Na,K-ATPase activity in the ciliary epithelium (Masferrer et al., 1990). The Na,K-ATPase of ciliary epithelium is widely believed to play a role in the mechanism of formation of aqueous humor (Cole, 1966 ; Davson, 1990). We have recently determined Na,K-ATPase activity in ciliary epithelium of the rabbit and have demonstrated that 12(R)-HETE does indeed inhibit the activity of this enzyme (Delamere et al., 1991). In the rabbit eye, HETEs are grouped together with a range of prostaglandins in the collection of arachidonic acid metabolites produced by both the cornea1 epithelium and the iris-ciliary body (Hurst et al., 1989 ; Davis, Dunn and Schwartzman, 1990). However, the iris-ciliary body contains a wide variety of cell types, and the arachidonic acid metabolites which are produced specifically by ciliary epithelium have not, to our knowledge, been documented. Since specific arachidonic acid metabolites might influence the ion transport function of the ciliary epithellum or may disrupt the blood-aqueous barrier (Eakins, 19 77) it is important to determine the pattern of metabolites that the epithelium can synthesize. For this reason, we isolated the ciliary epithelium from the rabbit eye and compared the profile of arachidonic acid metabolites produced by ciliary epithelium with that of metabolites produced by iris-ciliary body under similar experimental conditions.
0 1992 Academic Press Limited
K. L. KING
236
2. Materials and Methods Animals The eyes from Adult New Zealand White rabbits were used in these studies. The eyes were obtained from Pel Freeze Biologicals (Rogers, AR), shipped overnight on wet ice. The use of animals in this study conformed to .the Guide for Care and Use of Laboratory Animals (DHEW Publication NM 86-23). Chemicals [14C]Arachidonic acid, [3H]prostaglandin E, WU [3H]prostaglandin F,, (PGF,,), [3H]6 ketoprostaglandin F,, (Gk-PGF,,), [“Hjprostaglandm D, (PGD,), [3H]leukotriene B, (LTB,) were obtained from Du Pont-New England Nuclear (Boston, MA) and Amersham (Arlington Heights, IL). The unlabeled arachidonic acid metabolites 5-HETE, 12-HETE, HHT and thromboxane B, (TXB,) were obtained from Cayman Chemical (Ann Arbor, MI) and Biomol Research Lab., Inc. (Plymouth Meeting, PA). Organic solvents (liquid chromatography grade) and Silica1 Gel 60 thin layer chromatography plates, without fluorescent indicator, were obtained from EM Science (Cherry Hill, NJ). For gas chromatography/mass spectrometry experiacid ments, deuterated arachidonic (5,6,8,9,11,12,14,15-d,) was purchased from Cayman Chemical (Ann Arbor, MI). Pentafluorobenzyl bromide (PFB), methoxyamine hydrochloride (2% in pyridine, MOX) and trifluoroacetic anhydride (TFA anhydride) were purchased from Pierce Chemical Co. (Rockville, IL). All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI). Tissue Preparation
After the cornea was removed from the eye, the iris, ciliary body and lens were removed in one piece and transferred to a dish of ice cold 50 mM phosphate buffer, pH 75. Vitreous humor was removed by blotting on moist filter paper. A radial cut was made through the iris from the pupil to the periphery and the iris-ciliary body was then gently peeled away, leaving the ciliary epithelium attached to the lens zonules. The lens zonules were then cut in order to free the ciliary epithelium as a narrow strip of tissue. Microscopic examination of the ciliary epithelium showed that both cell layers remained joined and that it was free from contamination by ciliary process stroma or capillaries. This dissection technique has been described in detail by Jumblatt, Raphael and Jumblatt (1991). The tissue was divided into two parts: ciliary epithelium and the remaining iris-ciliary body. Arachidonic acid metabolism was examined using either the stripped ciliary epithelium or the iris-ciliary
ET AL.
body from which the epithelium samples had been removed. It should be noted, however, that not all of the ciliary epithelium was removed from the irisciliary body ; significant amounts of ciliary epithelium still remained attached to the ciliary processes of the ciliary body. To obtain sufficient tissue, ciliary epithelium samples were pooled from six eyes. The samples of ciliary epithelium or iris-ciliary body were homogenized in 500 ,ul of ice-cold phosphate buffer. The protein concentration of the tissue homogenate was determined by a Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA). Arachidonic acid metabolism by either ciliary epithelium or iris-ciliary body, was measured by adding 0.2 ,&i of 14C-labeled arachidonic acid (52 Ci mol-‘) to tubes containing 500 ,ul phosphate buffer and homogenized tissue at a final concentration of 0.6-0.8 mg protein per tube. The 6nal concentration of arachidonic acid was 3.4 x low6 M. The reaction mixture was incubated at 37°C for 45 min which allows sufficient time for detectable arachidonic acid metabolism to occur (Hurst et al., 1989; Williams et al., 1985). After incubation, 20 ,ul of 1 N HCl was added to each tube and the tubes placed on ice. To extract the arachidonic acid and its metabolites from the aqueous solution, 3 ml of ethyl acetate were added to each tube and mixed for 1 min before the tubes were centrifuged for 5 min at 3000 rpm. After centrifugation, 2.8 ml of the upper (organic) layer were removed and dried under a stream of nitrogen. The residue was resuspended in 20 yl of ethyl acetate and placed on a silica gel thin layer chromatography (TLC) plate. The arachidonic acid products were separated by running the TLC plates at 5°C in an A9 solvent system prepared from 25 : 50 : 50 : 10 trimethyl pentane, water, ethyl acetate and acetic acid by volume. In some experiments, we used a P solvent system containing 60 : 40 : 1 diethyl ether, petroleum ether and acetic acid (by volume). The TLC plate was then air-dried and the location of 14C-labeled arachidonic acid products was determined and quantified using an automated TLC plate scanner (Isomess IM-3016 Ray Test, USA Inc., McMurray, PA). TLC Standards
Standards were run on the TLC plate in lanes parallel with the biological samples. Radiolabelled standards were identified by scanning the plate. Nonlabeled standards were identified by developing the plate in the presence of iodine crystals and marking the location of the revealed bands. Gas Chromatography/Mass
Due to the dif6culty based strictly upon TLC confirm the formation thelium. For GC/MS
Spectrometry (GC/MS)
of identifying any compound mobilities, GC/MS was used to of 12-HETE by ciliary epistudies, homogenized ciliary
(A)
ARACHIDONIC
ACID
BY
METABOLISM
CILIARY
237
EPITHELIUM
2
I
soot
0
1020
3040~60708090100110120IM1401501~ lmml
$0 I? t
0 0
IO
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00
90
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I60
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210 0
(mm)
FIG. 1. Typical radio-thin-layer chromatography (TLC) scans of arachidonic acid metabolites synthesized by rabbit iris-ciliary body (A) and rabbit ciliary epithelium (B). Tissue homogenate (06-0.8 mg protein) was incubated in [‘*C]arachidonic acid for 45 min. Arachidonic acid metabolites were extracted and applied to TLC plates run using the A9 solvent system as described in Materials and Methods. The total dpm applied to the TLC plates was the same for both ciliary epithelium and iris-ciliary body. The inset to (A) shows the TLC scan of arachidonic acid metabolites synthesized by rabbit iris-ciliary body in the presence of 1 ,uM indomethacin. fndomethacin treatment caused no detectable change in the synthesis of arachidonic acid products by ciliary epithelium. PGE,, PGF,, 6k-PGF,,, and PGD, represent prostaglandins E,, F,,, 6 keto-F,, and D,, respectively; LTB, is leukotriene B, ; S-HETE and 12-HETE are S-and 12- hydroxyeicosatetranoic acid, respectively ; HHT is hydroxyheptadecatrienoic acid: TXB, is thromboxane B,; AA is arachidonic acid.
epithelium
was
incubated
for 45 min (as described
above) with unlabelled arachidonic acid (3.4 x 1O-6 M). In specified experiments, the homogenate was incubated with an equimolar mix of arachidonic acid and deuterated arachidonic acid. After the
been used for many years. The application of derivitization and GC/MS to eicosanoid analysis has recently been discussed in detail by Blair (1990). Authentic standard 12-HETE, or ethyl acetate extracts of tissue homogenates were taken for analysis. Water was
incubation,
removed
authentic ization.
samples
of the
reaction
mixture
12-HETE were analysed following
or
derivit-
Derivitization. This approach to GC/MS analysis has
using sodium sulfate and 10 ~1 of a 1 g 1-l
solution of 12-hydroxystearic acid was added to each sample. The samples were then transferred to silanized vials and evaporated to dryness under a stream of dry nitrogen at room temperature. The residues were
238 reconstituted in 100 ~1 of dry acetonitrile, then 50 ~1 each of NJV-diisopropylethylamine and PFB were added, in that order, to form the PFB esters of carboxylic acids. Following a 30-min incubation at room temperature, the samples were evaporated to dryness under a stream of dry nitrogen at room temperature. To each vial were added 50 ,ul of MOX to form methoximes of carbonyls. After 1 hr at room temperature, samples were dried as described above, and 50 ~1 each of TFA anhydride and acetonitrile were added. After a final 20-min incubation at room temperature and drying as described, the residue was dissolved in 5050 (by volume) dichloromethane : ethyl acetate. These yellow extracts were further purified by application to a l-ml column of prewashed silica gel, then elution using the same solvent system. GC/MS analysis. Samples were subjected to GC/MS analysis using a HP-5890A GC (Hewlett Packard, Palo Alto, CA) fitted with a 10 m x 0.18 mm DB-1 fused silica bonded capillary column with a iilm thickness of 0.4 pm (J & W Scientific, Folsom, CA). Grade V helium was the carrier gas. Temperature programmed analyses were performed. The oven temperature was held at 80°C for 2 min, then ramped to 275°C at a rate of 10°C min-‘. The column effluent was directed into a HP-5970 Mass-Selective Detector (Hewlett Packard), where the effluent was subjected to 70 eV electron ionization and positive ion analysis. The quadrupole was programmed to scan from m/z = 800 to 35 at the rate of 0.6 Hz.
K. L. KING
ET AL.
by iris-ciliary body. Control TLC scans of 14C-labeled arachidonic acid alone showed the labeled compound to be pure and to migrate as one single peak. In these studies we used tissues isolated from commercially available rabbit eyes shipped overnight on wet ice. In control experiments, we established that tissues isolated from freshly removed rabbit eyes showed essentially the same pattern of arachidonic acid metabolism as tissues isolated from the chilled rabbit eyes. The amount of each arachidonic acid metabolite was determined from the radioactivity associated with its peak on the TLC scan. Figure 2. summarizes the data obtained from several radio-chromatograms. The arachidonic acid metabolites are shown grouped into two categories : cyclooxygenase metabolites ; and SHBTE and 12-HETE. There was a clear difference between the pattern of arachidonic acid metabolites produced by the iris-ciliary body and the pattern produced by the ciliary epithelium. The group of compounds comprising 5-HETE and 12-HETE constituted approximately 66 % of the total arachidonic acid metabolites produced by the ciliary epithelium; in contrast this group of metabolites comprised less than 20 % of the arachidonic acid metabolites generated by iris-ciliary body. The iris-ciliary body appeared to preferentially generate the group of compounds made up by Gk-PGF,,, PGE,, PGD,, PGF,,, TXB, and HHT; these products were not prominent in the TLC scans of arachidonic acid metabolites generated by ciliary epithelium. Under conditions of our experiments, the overall conversion of arachidonic acid by the ciliary epithelium was less than the conversion observed with
3. Results TLC Analysis
The iris with attached ciliary body was removed from the rabbit eye and then divided into two components, pure ciliary epithelium and iris-ciliary body. Metabolism of 14C-labeled arachidonic acid was examined in both tissues. The labeled metabolites, separated by TLC using the A9 solvent system, are shown in Figs l(A) and (B). Arachidonic acid metabolites produced by the iris-ciliary body comigrated on the TLC plates with Gk-PGF,,, which is a stable metabolite of prostacyclin, PGF,,, PGEJI’XB,, PGD,, 5-HBTE, HHT and 12-HETE [Fig. l(A)]. When indomethacin (1 PM) was added 15 min prior to the addition of arachidonic acid, the products which comigrated with prostaglandins were not detectable [Fig. l(A) inset]. The ciliary epithelium exhibited a diierent pattern of arachidonic acid products. Metabolites produced by the ciliary epithelium co-migrated with 5HETE and 12-HETE [Fig. l(B)]. Some material from the ciliary epithelium samples did co-migrate with PGF,,, PGEJI’XB, and PGD,, but the amount of these products was markedly less than the amount generated
Iris-ciliary
bW
Ciliary eplthehum
FIG. 2. 14C-labeledarachidonic acid metabolites generated by rabbit iris-ciliary body and rabbit ciliary epithelium. The arachidonic acid metabolites are grouped into two categories: cyclooxygenase products (m): and 5- and 12-HETE (0). The data of the mean of three separate experiments with the standard error shown as a vertical bar.
ARACHIDONIC
ACID
METABOLISM
BY CILIARY
EPITHELIUM
239
aa
250
200
VI 150 E ” 100
50
0 0
IO
20
30
40
50
60
70
60
90
100
II0
120
130
I40
I50
I60
I70
I80
190
200
(mm)
FIG. 3. Typical radio-thin-layer chromatography (TLC) scan of arachidonic acid metabolites synthesized by rabbit ciliary epithelium. Tissue homogenate (0.6 mg protein) was incubated in [W]arachidonic acid for 45 min. Arachidonic acid metaholites were extracted and applied to TLC plates run using the P solvent system as described in Materials and Methods.
(A) IOO-
181 CPFBI+
75 263 50 261 25:
265
I I 150
200
.
.I
I. I I 250
II I,
283
L. . . 3 0
m/r
FIG. 4. Partial mass spectra are shown for the derivative of authentic 12-HBTE (A), the same derivative of 12-HETE from ciliary epithelium (B) and the derivative formed by ciliary epithelium from 12-HBTl3 produced from deuterated arachidonic acid (C). The vertical axis represents relative ion current intensity. The horizontal axis represents the mass/charge (m/z) ratio for each ion. an equivalent amount of iris-ciliary body tissue (determined on the basis of total protein). The ciliary epithelium metabolized 10.7%f6-5 (s.E.M.) of the available arachidonic acid while the iris-ciliary body
metabolized 459%f 10.2 (s.E.M.). To better identify HETE production by ciliary epithelium, we also separated the metabolites by TLC using a different solvent system, P, which separated less polar arachi-
K. L. KING
240
donic acid metabolites (Fig. 3). This separated more clearly the material which co-migrated with S-HETE and 12-HETE. Gas Chromatography/Mass Analysis
Spectrometry (GC/MS)
The arachidonic acid metabolites generated by ciliary epithelium were analysed by GC/MS to co&m the production of 12-HETE. The derivative of the internal standard for analysis, 1-periluorobenzyl-12trifluoroacetyl-octadecanoate had an absolute retention time of 22.1 min. The corresponding derivative of authentic 12-HETE had a retention index (retention time relative to that of the standard) of 0.9697. A compound with a retention index of 0.9691 and the same mass spectrum was found in extracts from ciliary epithelium which had been incubated with arachidonic acid [Figs 4(A) and (B)]. Analysis of extracts of ciliary epithelium which had been fortified with an equimolar mixture of arachidonic acid and d,arachidonic acid showed a partially resolved doublet of peaks, one corresponding to the deuterated product [Fig. 4(C)]. 4. Discussion
The rabbit ciliary epithelium was observed to be capable of metabolizing ‘*C-labeled arachidonic acid, although the net amount of arachidonic acid converted by the ciliary epithelium was lower than that of the iris-ciliary body. Ciliary epithelium produced substantial amounts of arachidonic acid metabolites that co-migrated with S-HETE and 12-HETE in two diierent TLC solvent systems. Surprisingly, the ciliary epithelium produced only small amounts of cyclooxygenase products such as PGF,,, PGE,, PGD, and Gk-PGF,,. 12-HETE production by ciliary epithelium was further supported by co-migration under GC conditions and by MS analysis since it is not possible to identify a compound based strictly upon TLC mobility. Following methodology described in detail by Blair (1990), a derivatization scheme was employed to provide more volatile and more stable analytes, makiig PFB esters of carboxyl groups, methoximes of carbonyls (especially enolizable keto functions) and finally, TFA esters of hydroxyl groups. This scheme also introduces two ‘reporter’ groups (PFB and TFA) which consistently yield ions of m/z = 181 (PFB) and 69 (CF, from TFA). The lower m/z portions of the spectra simply showed the characteristics of unsaturated fatty acids and TFA esters. The production of deuterium-labeled 12-HETE demonstrated that this was generated from the exogenous arachidonic acid. The observed metabolites of arachidonic acid by irisciliary body were diierent from that observed for ciliary epithelium. Iris-ciliary body produced large amounts of cyclooxygenase products such as PGF,,
ET AL.
and PGD,. Generation of these products was suppressed by the cyclooxygenase inhibitor indomethacin. While iris-ciliary body did produce measurable quantities of compounds that co-migrated with 5-HETE and 12-HETE, the amount of these compounds was small relative to the amount of cyclooxygenase products that were formed. The pattern of arachidonic acid metabolism by the iris-ciliary body was similar to that reported by earlier investigators (Hurst et al., 1989). It is noteworthy that ciliary epithelium appears capable of metabolizing arachidonic acid to produce 12-HETE. From these experiments we cannot tell whether the epithelium produces 12(R)-HETE. 12(S)HETE or both. 12(R)-HETE, an Na,K-ATPase inhibitor is capable of lowering intraocular pressure ; 12(S)HETE does not cause such changes (Masferrer et al., 1990 ; Delamere et al., 1991). Masferrer et al. (1990) have speculated that 12(R)-HETE might be an endogenous modulator of Na,K-ATPase in the ciliary epithelium. However, it should be recognized that the HETE family of compounds has a wide range variety of biological effects, many of them concerned with the inflammatory response. For example, both 12(S)-HETE and 12(R)-HETE are recognised neutrophil attractants (Palmer et al., 1980; Wiggins, Jafri and Proia, 19 90). Other arachidonic acid metabolites, such as HHT, also exhibit chemotactic activity for inflammatory cells (Goetzl and Gorman, 1978). In addition, arachidonic acid metabolism itself may be altered by HETES: SHETE appears to be able to stimulate prostaglandin production by cultured MDCK cells (Gordon et al., 1989). The finding that ciliary epitheIium poorly metabolizes arachidonic acid into cyclooxygenase products may be important for the interpretation of prostaglandin effects upon the eye. Prostaglandins are well known for their contribution to inflammatory reactions in the eye (Bito, Nichols and Baroody. 19 82 ; Bhattacherjee, 1989 ; Kulkarni and Srinivasan, 19 89) and certain prostaglandins PGI, (prostacyclin) and PGE, have been shown to cause breakdown of the blood-aqueous barrier (Eakins, 19 7 7 ; Desantis and Sallee, 1989), possibly by altering the permeability of the ciliary epithelial barrier to large molecules (Vegge, Neufeld and Sears, 1975). Furthermore, some cyclooxygenase products are able to lower intraocular pressure (Crawford and Kaufman, 1987; Gabelt and Kaufman, 1989). The low efficiency of prostaglandin production which we observed in ciliary epithelium suggests that prostaglandin-mediated changes in the blood-aqueous barrier at the ciliary epithelium are likely to be caused by prostaglandins produced by tissues outside the epithelium. Acknowledgements This work was supported by USPHS Research Grants .No. EY06915 and EYO6918, the Kentucky Lions Eye Foun-
dation, and an unrestricted grant from Research to Prevent Blindness, Inc. We wish to thank Drs Parimal Bhattacherjee,
ARACHIDONIC
ACID
METABOLISM
BY CILIARY
EPITHELIUM
Prasad Kulkarni and Christopher A. Paterson for helpful discussion during the course of this work.
References Bazan, H. E. P. (1989). The synthesis and effects of eicosanoids in avascular ocular tissues. In The OcuZurEffects of ProstagZandins and Other Eicosanoids. (Eds Bito, L. 2. and Stjernschantz, J.). Pp. 73-84. Liss: New York. Bhattacherjee. P. (1989). The role of arachidonic metabolite in ocular inflammation. In The Ocular Effects of Prostaglandins andOtherEicosanoids. (EdsBito, L. 2. and Stjernschantz,J.). Pp. 211-27. Liss:New York. Bito, L. Z., Nichols, R. R. and Baroody, R. A. (1982). A comparisonof the miotic and inflammatory effectsof biologically active polypeptidesand prostaglandinE, on the rabbit eye. Exp. Eye Res. 34, 325-37. Blair, I. A. (1990). Electron-capturenegative-ion chemical ionization massspectrometryof lipid mediators.Methods Enzymol. 187, 13-23. Cole, D. F. (1966). Aqueous humor formation. Dot. Ophthalmol.21, 116-238. Crawford, K. and Kaufman, P. L. (1987). Pilocarpine antagonizes prostaglandin F,,-induced ocular hypotensionin monkeys.Arch. Ophthalmol. 105, 1112-6. Davis, K. L., Dunn, M. W. and Schwartzman, M. L. (1990). Hormonal stimulation of 12(R)-HETE, a cytochrome P450 arachidonicacid metabolitein the rabbit cornea. Curr. Eye Res. 9, 661-7. Davson, H. (1990). Aqueous humor and the intraocular pressure.In Physiology of the Eye. (Ed. Davson,H.). Pp. 9-81. AcademicPress:New York. Delamere,N. A., Socci, R. R., Bhattachergee,P. and King K. L. (1991). Studies on the intraocular pressurelowering effect of 12 (R)-Hydroxyeicosatetraenoicacid in the rabbit. Invest. OpthalmoI. Vis. Sci. 32, 25114. Desantis.L. L.. Jr. and Sallee,V. L. (1989). Comparisonof the effectsof prostaglandinsand their esterson bloodaqueousbarrier integrity and intraocular pressurein rabbits.In The Ocular Effects of Prostaglandins and Other Eicosanoids. (EdsBito. L Z. and Stjernschantz,7.). Pp. 379-86. Liss:New York. Eakins,K. E. ( 1977). Prostaglandinand non-prostaglandin mediatedbreakdownof the blood-aqueous barrier. Exp. Eye Res. 25, 483-98.
Escalante, B.. Falck, J. R., Yadagiri, P., Sun, L. and Schwartzman. M. L. (1988). 19(S)-hydroxyeicosatetraenoic acid is a potent stimulator of renal Na+-K+ATPase. Biochem. Biophys. Res. Commun. 152, 1269-74.
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Gabelt,B. T. and Kaufman, P. L. (1989). ProstaglandinF,, increases uveoscleral outflow in the cynomolgus monkey. Exp. EyeRes.49, 389402. Goetzl, E.J. and Gorman, R. R. (1978). Chemotacticand chemokinetic stimulation of human eosinophil and neutrophil polymorphonuclear leukocytes by 12-Lhydroxy- 5,8,10-heptadectrienoic acid (HHT). J. Immunoi. 2, 526-31. Gordon,J. A., Figard, P H., Quinby, G. E. and Spector,A. A. (1989). 5-HETE:uptake, distribution, and metabolism in MDCK cells.Am. J. Physiol. 256, Cl-ClO. Hurst, J. S., Paterson,C. A., Bhattacherjee.P. and Pierce, W. M. (1989). Effectsof ebselenon arachidonatemetabolism by ocular and non-ocular tissues. Biochem. Pharmacol. 38, 3357-63.
Jumblatt, M. M., Raphael,B. and Jumblatt. J. E. (1991). A simplemethod for the isolation of ciliary epithelium. Exp. Eye Res. 52, 229-32.
Kulkarni, P. S., Fleisher,L. and Srinivasan, B. D. (1984). The synthesisof cyclooxygenaseproducts in ocular tissuesof various species.Curr. Eye Res. 3, 447-52. Kulkarni, P. S.andSrinivasan.B. D. (1989). Cyclooxygenase and lipoxygenase pathways in anterior uvea and conjunctiva. In The Ocular Eflectsof Prostaglandins and Other Eicosanoids. (EdsBito. L. Z. and Stjernschantz,J.). Pp. 39-52. Liss:New York. Masferrer, J. L., Dunn, M. W. and Schwartzman, M. L. (1990). 12(R)-hydroxyeicosatetraenoicacid, an endogenous cornea1arachidonate metabolite, lowers intraocular pressurein rabbits. Invest. Ophthnlmol. Vis. Sci. 31, 535-9. Palmer, R. M. J.. Stepney, R. J., Higgs. G. A. and Eakins. K. E. (1980). Chemokineticactivity of arachidonicacid lipoxygenaseproductson ieucocytesof differentspecies. Prostaglandins 20, 411-8. Schwartzman, M. L., Balazy, M., Masferrer. J., Abraham, N. G., McGiff, J. C. and Murphy, R. C. (1987). 12(R)hydroxyeicosatetraenoic acid: a cytochrome P4 50dependentarachidonatemetabolite that inhibits Na’K+-ATPasein the cornea. Proc. Natl. Acad. Sci. U.S.A. 84,
8125-9.
Vegge, T.. Neufeld, A. H. and Sears, M. L. (19 75). Morphology of the breakdownof the blood-aqueous barrier in the ciliary processesof the rabbit eye after prostaglandin E,. Invest. Ophthnlmol. Vis. Sci. 14. 33-6. Wiggins, R. E., Jafri, M. S. and Proia, A. D. (1990). 12(S)hydroxy-5,8,10,14-eicosatetraenoic acid is a more potent neutrophil chemoattractant than the 12(R) epimerin the rat cornea.Prostuglundins 40, 131-41. Williams,R. N., Delamere,N. A. and Paterson,C. A. (1985). Generationof lipoxygenaseproducts in the avascular tissuesof the eye. Exp. Eye Res. 41, 73 3-8.
55, 235-241
Metabolism
of Arachidonic
KARA L. KING”,
Acid Epithelium
by Isolated
NICHOLAS A. DELAMERET, STEPHEN AND WILLIAM M. PIERCE, JR.”
Rabbit
Ciliary
C.CSUKAS
Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Research Institute and aDepartment of Pharmacology, and Toxicology, University of Louisville School of Medicine, Louisville, KY 40292, U.S.A. (Received Chicago 22 March 1991 and accepted in revised form 21 November 7991) We examined the ability of rabbit ciliary epithelium to metabolize arachidonic acid in vitro. The epithelium was homogenizedand incubatedwith l*Clabeled arachidonicacid. **C-labeledmetabolites were extracted and then separatedby thin layer chromatography. The range of arachidonic acid metabolitessynthesizedby ciliary epitheliumwas comparedto the metabolitesgeneratedby rabbit irisciliary body. Ciliary epitheliumproducedsubstantialamountsof arachidonic acid metabolitesthat comigrated with 5-HRTFand 12-HRTE.Authenticity of the 12-HRTEproducedby ciliary epithelium was confirmed by gas chromatography/massspectrometry. The ciliary epithelium generatedonly small amountsof the cyclooxygenaseproducts,PGF,,, PGE,,PGD, and Gk-PGP,,.In contrast, the iris-ciliary body producedlarge amountsof cyclooxygenaseproducts such as PGF,, and PGD,. The ability of the ciliary epithelium to generate 12-HETEis noteworthy since 12(R)-HRTFis known to be capableof lowering intraocular pressure. Key words:ciliary epithelium; iris-ciliary body: rabbit; arachidonicacid: HETES;prostaglandins.
1. Introduction Arachidonic acid can be metabolized by three pathways : cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase. The metabolites of arachidonic acid can vary from one tissue to another, probably due to the different prominence of each specific metabolic pathway within diierent cell types. For example, the cornea and iris both appear capable of metabolizing a substantial amount of arachidonic acid by the lipoxygenase pathway (Kulkarni, Fleisher and Srinivasan, 1984; Hurst et al., 1989) while lipoxygenase activity in the lens is much lower (see Bazan, 1989). In fact, Williams, Delamere and Paterson (1985) failed to detect the generation of lipoxygenase products from exogenous arachidonic acid by either rabbit or frog lens. Arachidonic acid metabolites are well known for their role in inflammatory
responses of the eye
(Bhattacherjee, 1989). Recently, one particular arachidonic acid product, 12(R)-hydroxyeicosatetraenoic acid [ 12(R)-HETE] has received attention because it can lower intraocular pressure (Masferrer, Dunn and Schwartzman, 1990). Since 12(R)-HETE can inhibit Na,K-ATPase activity in cornea and other tissues (Schwartzman et al., 198 7 ; Escalante et al., 1988) it has been suggested that the * Present address : Discovery Research, Allergan Pharmaceuticals 2525 Dupont Drive, Irvine, CA 92715, U.S.A. t For reprint requests and correspondence at: Department of Ophthalmology and Visual Sciences, University of Louisville, Louisville. KY 40292, U.S.A.
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TOP-lowering effect of 12(R)-HETE might be related to the inhibition of Na,K-ATPase activity in the ciliary epithelium (Masferrer et al., 1990). The Na,K-ATPase of ciliary epithelium is widely believed to play a role in the mechanism of formation of aqueous humor (Cole, 1966 ; Davson, 1990). We have recently determined Na,K-ATPase activity in ciliary epithelium of the rabbit and have demonstrated that 12(R)-HETE does indeed inhibit the activity of this enzyme (Delamere et al., 1991). In the rabbit eye, HETEs are grouped together with a range of prostaglandins in the collection of arachidonic acid metabolites produced by both the cornea1 epithelium and the iris-ciliary body (Hurst et al., 1989 ; Davis, Dunn and Schwartzman, 1990). However, the iris-ciliary body contains a wide variety of cell types, and the arachidonic acid metabolites which are produced specifically by ciliary epithelium have not, to our knowledge, been documented. Since specific arachidonic acid metabolites might influence the ion transport function of the ciliary epithellum or may disrupt the blood-aqueous barrier (Eakins, 19 77) it is important to determine the pattern of metabolites that the epithelium can synthesize. For this reason, we isolated the ciliary epithelium from the rabbit eye and compared the profile of arachidonic acid metabolites produced by ciliary epithelium with that of metabolites produced by iris-ciliary body under similar experimental conditions.
0 1992 Academic Press Limited
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2. Materials and Methods Animals The eyes from Adult New Zealand White rabbits were used in these studies. The eyes were obtained from Pel Freeze Biologicals (Rogers, AR), shipped overnight on wet ice. The use of animals in this study conformed to .the Guide for Care and Use of Laboratory Animals (DHEW Publication NM 86-23). Chemicals [14C]Arachidonic acid, [3H]prostaglandin E, WU [3H]prostaglandin F,, (PGF,,), [3H]6 ketoprostaglandin F,, (Gk-PGF,,), [“Hjprostaglandm D, (PGD,), [3H]leukotriene B, (LTB,) were obtained from Du Pont-New England Nuclear (Boston, MA) and Amersham (Arlington Heights, IL). The unlabeled arachidonic acid metabolites 5-HETE, 12-HETE, HHT and thromboxane B, (TXB,) were obtained from Cayman Chemical (Ann Arbor, MI) and Biomol Research Lab., Inc. (Plymouth Meeting, PA). Organic solvents (liquid chromatography grade) and Silica1 Gel 60 thin layer chromatography plates, without fluorescent indicator, were obtained from EM Science (Cherry Hill, NJ). For gas chromatography/mass spectrometry experiacid ments, deuterated arachidonic (5,6,8,9,11,12,14,15-d,) was purchased from Cayman Chemical (Ann Arbor, MI). Pentafluorobenzyl bromide (PFB), methoxyamine hydrochloride (2% in pyridine, MOX) and trifluoroacetic anhydride (TFA anhydride) were purchased from Pierce Chemical Co. (Rockville, IL). All other chemicals were purchased from Aldrich Chemical Co. (Milwaukee, WI). Tissue Preparation
After the cornea was removed from the eye, the iris, ciliary body and lens were removed in one piece and transferred to a dish of ice cold 50 mM phosphate buffer, pH 75. Vitreous humor was removed by blotting on moist filter paper. A radial cut was made through the iris from the pupil to the periphery and the iris-ciliary body was then gently peeled away, leaving the ciliary epithelium attached to the lens zonules. The lens zonules were then cut in order to free the ciliary epithelium as a narrow strip of tissue. Microscopic examination of the ciliary epithelium showed that both cell layers remained joined and that it was free from contamination by ciliary process stroma or capillaries. This dissection technique has been described in detail by Jumblatt, Raphael and Jumblatt (1991). The tissue was divided into two parts: ciliary epithelium and the remaining iris-ciliary body. Arachidonic acid metabolism was examined using either the stripped ciliary epithelium or the iris-ciliary
ET AL.
body from which the epithelium samples had been removed. It should be noted, however, that not all of the ciliary epithelium was removed from the irisciliary body ; significant amounts of ciliary epithelium still remained attached to the ciliary processes of the ciliary body. To obtain sufficient tissue, ciliary epithelium samples were pooled from six eyes. The samples of ciliary epithelium or iris-ciliary body were homogenized in 500 ,ul of ice-cold phosphate buffer. The protein concentration of the tissue homogenate was determined by a Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA). Arachidonic acid metabolism by either ciliary epithelium or iris-ciliary body, was measured by adding 0.2 ,&i of 14C-labeled arachidonic acid (52 Ci mol-‘) to tubes containing 500 ,ul phosphate buffer and homogenized tissue at a final concentration of 0.6-0.8 mg protein per tube. The 6nal concentration of arachidonic acid was 3.4 x low6 M. The reaction mixture was incubated at 37°C for 45 min which allows sufficient time for detectable arachidonic acid metabolism to occur (Hurst et al., 1989; Williams et al., 1985). After incubation, 20 ,ul of 1 N HCl was added to each tube and the tubes placed on ice. To extract the arachidonic acid and its metabolites from the aqueous solution, 3 ml of ethyl acetate were added to each tube and mixed for 1 min before the tubes were centrifuged for 5 min at 3000 rpm. After centrifugation, 2.8 ml of the upper (organic) layer were removed and dried under a stream of nitrogen. The residue was resuspended in 20 yl of ethyl acetate and placed on a silica gel thin layer chromatography (TLC) plate. The arachidonic acid products were separated by running the TLC plates at 5°C in an A9 solvent system prepared from 25 : 50 : 50 : 10 trimethyl pentane, water, ethyl acetate and acetic acid by volume. In some experiments, we used a P solvent system containing 60 : 40 : 1 diethyl ether, petroleum ether and acetic acid (by volume). The TLC plate was then air-dried and the location of 14C-labeled arachidonic acid products was determined and quantified using an automated TLC plate scanner (Isomess IM-3016 Ray Test, USA Inc., McMurray, PA). TLC Standards
Standards were run on the TLC plate in lanes parallel with the biological samples. Radiolabelled standards were identified by scanning the plate. Nonlabeled standards were identified by developing the plate in the presence of iodine crystals and marking the location of the revealed bands. Gas Chromatography/Mass
Due to the dif6culty based strictly upon TLC confirm the formation thelium. For GC/MS
Spectrometry (GC/MS)
of identifying any compound mobilities, GC/MS was used to of 12-HETE by ciliary epistudies, homogenized ciliary
(A)
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FIG. 1. Typical radio-thin-layer chromatography (TLC) scans of arachidonic acid metabolites synthesized by rabbit iris-ciliary body (A) and rabbit ciliary epithelium (B). Tissue homogenate (06-0.8 mg protein) was incubated in [‘*C]arachidonic acid for 45 min. Arachidonic acid metabolites were extracted and applied to TLC plates run using the A9 solvent system as described in Materials and Methods. The total dpm applied to the TLC plates was the same for both ciliary epithelium and iris-ciliary body. The inset to (A) shows the TLC scan of arachidonic acid metabolites synthesized by rabbit iris-ciliary body in the presence of 1 ,uM indomethacin. fndomethacin treatment caused no detectable change in the synthesis of arachidonic acid products by ciliary epithelium. PGE,, PGF,, 6k-PGF,,, and PGD, represent prostaglandins E,, F,,, 6 keto-F,, and D,, respectively; LTB, is leukotriene B, ; S-HETE and 12-HETE are S-and 12- hydroxyeicosatetranoic acid, respectively ; HHT is hydroxyheptadecatrienoic acid: TXB, is thromboxane B,; AA is arachidonic acid.
epithelium
was
incubated
for 45 min (as described
above) with unlabelled arachidonic acid (3.4 x 1O-6 M). In specified experiments, the homogenate was incubated with an equimolar mix of arachidonic acid and deuterated arachidonic acid. After the
been used for many years. The application of derivitization and GC/MS to eicosanoid analysis has recently been discussed in detail by Blair (1990). Authentic standard 12-HETE, or ethyl acetate extracts of tissue homogenates were taken for analysis. Water was
incubation,
removed
authentic ization.
samples
of the
reaction
mixture
12-HETE were analysed following
or
derivit-
Derivitization. This approach to GC/MS analysis has
using sodium sulfate and 10 ~1 of a 1 g 1-l
solution of 12-hydroxystearic acid was added to each sample. The samples were then transferred to silanized vials and evaporated to dryness under a stream of dry nitrogen at room temperature. The residues were
238 reconstituted in 100 ~1 of dry acetonitrile, then 50 ~1 each of NJV-diisopropylethylamine and PFB were added, in that order, to form the PFB esters of carboxylic acids. Following a 30-min incubation at room temperature, the samples were evaporated to dryness under a stream of dry nitrogen at room temperature. To each vial were added 50 ,ul of MOX to form methoximes of carbonyls. After 1 hr at room temperature, samples were dried as described above, and 50 ~1 each of TFA anhydride and acetonitrile were added. After a final 20-min incubation at room temperature and drying as described, the residue was dissolved in 5050 (by volume) dichloromethane : ethyl acetate. These yellow extracts were further purified by application to a l-ml column of prewashed silica gel, then elution using the same solvent system. GC/MS analysis. Samples were subjected to GC/MS analysis using a HP-5890A GC (Hewlett Packard, Palo Alto, CA) fitted with a 10 m x 0.18 mm DB-1 fused silica bonded capillary column with a iilm thickness of 0.4 pm (J & W Scientific, Folsom, CA). Grade V helium was the carrier gas. Temperature programmed analyses were performed. The oven temperature was held at 80°C for 2 min, then ramped to 275°C at a rate of 10°C min-‘. The column effluent was directed into a HP-5970 Mass-Selective Detector (Hewlett Packard), where the effluent was subjected to 70 eV electron ionization and positive ion analysis. The quadrupole was programmed to scan from m/z = 800 to 35 at the rate of 0.6 Hz.
K. L. KING
ET AL.
by iris-ciliary body. Control TLC scans of 14C-labeled arachidonic acid alone showed the labeled compound to be pure and to migrate as one single peak. In these studies we used tissues isolated from commercially available rabbit eyes shipped overnight on wet ice. In control experiments, we established that tissues isolated from freshly removed rabbit eyes showed essentially the same pattern of arachidonic acid metabolism as tissues isolated from the chilled rabbit eyes. The amount of each arachidonic acid metabolite was determined from the radioactivity associated with its peak on the TLC scan. Figure 2. summarizes the data obtained from several radio-chromatograms. The arachidonic acid metabolites are shown grouped into two categories : cyclooxygenase metabolites ; and SHBTE and 12-HETE. There was a clear difference between the pattern of arachidonic acid metabolites produced by the iris-ciliary body and the pattern produced by the ciliary epithelium. The group of compounds comprising 5-HETE and 12-HETE constituted approximately 66 % of the total arachidonic acid metabolites produced by the ciliary epithelium; in contrast this group of metabolites comprised less than 20 % of the arachidonic acid metabolites generated by iris-ciliary body. The iris-ciliary body appeared to preferentially generate the group of compounds made up by Gk-PGF,,, PGE,, PGD,, PGF,,, TXB, and HHT; these products were not prominent in the TLC scans of arachidonic acid metabolites generated by ciliary epithelium. Under conditions of our experiments, the overall conversion of arachidonic acid by the ciliary epithelium was less than the conversion observed with
3. Results TLC Analysis
The iris with attached ciliary body was removed from the rabbit eye and then divided into two components, pure ciliary epithelium and iris-ciliary body. Metabolism of 14C-labeled arachidonic acid was examined in both tissues. The labeled metabolites, separated by TLC using the A9 solvent system, are shown in Figs l(A) and (B). Arachidonic acid metabolites produced by the iris-ciliary body comigrated on the TLC plates with Gk-PGF,,, which is a stable metabolite of prostacyclin, PGF,,, PGEJI’XB,, PGD,, 5-HBTE, HHT and 12-HETE [Fig. l(A)]. When indomethacin (1 PM) was added 15 min prior to the addition of arachidonic acid, the products which comigrated with prostaglandins were not detectable [Fig. l(A) inset]. The ciliary epithelium exhibited a diierent pattern of arachidonic acid products. Metabolites produced by the ciliary epithelium co-migrated with 5HETE and 12-HETE [Fig. l(B)]. Some material from the ciliary epithelium samples did co-migrate with PGF,,, PGEJI’XB, and PGD,, but the amount of these products was markedly less than the amount generated
Iris-ciliary
bW
Ciliary eplthehum
FIG. 2. 14C-labeledarachidonic acid metabolites generated by rabbit iris-ciliary body and rabbit ciliary epithelium. The arachidonic acid metabolites are grouped into two categories: cyclooxygenase products (m): and 5- and 12-HETE (0). The data of the mean of three separate experiments with the standard error shown as a vertical bar.
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FIG. 3. Typical radio-thin-layer chromatography (TLC) scan of arachidonic acid metabolites synthesized by rabbit ciliary epithelium. Tissue homogenate (0.6 mg protein) was incubated in [W]arachidonic acid for 45 min. Arachidonic acid metaholites were extracted and applied to TLC plates run using the P solvent system as described in Materials and Methods.
(A) IOO-
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FIG. 4. Partial mass spectra are shown for the derivative of authentic 12-HBTE (A), the same derivative of 12-HETE from ciliary epithelium (B) and the derivative formed by ciliary epithelium from 12-HBTl3 produced from deuterated arachidonic acid (C). The vertical axis represents relative ion current intensity. The horizontal axis represents the mass/charge (m/z) ratio for each ion. an equivalent amount of iris-ciliary body tissue (determined on the basis of total protein). The ciliary epithelium metabolized 10.7%f6-5 (s.E.M.) of the available arachidonic acid while the iris-ciliary body
metabolized 459%f 10.2 (s.E.M.). To better identify HETE production by ciliary epithelium, we also separated the metabolites by TLC using a different solvent system, P, which separated less polar arachi-
K. L. KING
240
donic acid metabolites (Fig. 3). This separated more clearly the material which co-migrated with S-HETE and 12-HETE. Gas Chromatography/Mass Analysis
Spectrometry (GC/MS)
The arachidonic acid metabolites generated by ciliary epithelium were analysed by GC/MS to co&m the production of 12-HETE. The derivative of the internal standard for analysis, 1-periluorobenzyl-12trifluoroacetyl-octadecanoate had an absolute retention time of 22.1 min. The corresponding derivative of authentic 12-HETE had a retention index (retention time relative to that of the standard) of 0.9697. A compound with a retention index of 0.9691 and the same mass spectrum was found in extracts from ciliary epithelium which had been incubated with arachidonic acid [Figs 4(A) and (B)]. Analysis of extracts of ciliary epithelium which had been fortified with an equimolar mixture of arachidonic acid and d,arachidonic acid showed a partially resolved doublet of peaks, one corresponding to the deuterated product [Fig. 4(C)]. 4. Discussion
The rabbit ciliary epithelium was observed to be capable of metabolizing ‘*C-labeled arachidonic acid, although the net amount of arachidonic acid converted by the ciliary epithelium was lower than that of the iris-ciliary body. Ciliary epithelium produced substantial amounts of arachidonic acid metabolites that co-migrated with S-HETE and 12-HETE in two diierent TLC solvent systems. Surprisingly, the ciliary epithelium produced only small amounts of cyclooxygenase products such as PGF,,, PGE,, PGD, and Gk-PGF,,. 12-HETE production by ciliary epithelium was further supported by co-migration under GC conditions and by MS analysis since it is not possible to identify a compound based strictly upon TLC mobility. Following methodology described in detail by Blair (1990), a derivatization scheme was employed to provide more volatile and more stable analytes, makiig PFB esters of carboxyl groups, methoximes of carbonyls (especially enolizable keto functions) and finally, TFA esters of hydroxyl groups. This scheme also introduces two ‘reporter’ groups (PFB and TFA) which consistently yield ions of m/z = 181 (PFB) and 69 (CF, from TFA). The lower m/z portions of the spectra simply showed the characteristics of unsaturated fatty acids and TFA esters. The production of deuterium-labeled 12-HETE demonstrated that this was generated from the exogenous arachidonic acid. The observed metabolites of arachidonic acid by irisciliary body were diierent from that observed for ciliary epithelium. Iris-ciliary body produced large amounts of cyclooxygenase products such as PGF,,
ET AL.
and PGD,. Generation of these products was suppressed by the cyclooxygenase inhibitor indomethacin. While iris-ciliary body did produce measurable quantities of compounds that co-migrated with 5-HETE and 12-HETE, the amount of these compounds was small relative to the amount of cyclooxygenase products that were formed. The pattern of arachidonic acid metabolism by the iris-ciliary body was similar to that reported by earlier investigators (Hurst et al., 1989). It is noteworthy that ciliary epithelium appears capable of metabolizing arachidonic acid to produce 12-HETE. From these experiments we cannot tell whether the epithelium produces 12(R)-HETE. 12(S)HETE or both. 12(R)-HETE, an Na,K-ATPase inhibitor is capable of lowering intraocular pressure ; 12(S)HETE does not cause such changes (Masferrer et al., 1990 ; Delamere et al., 1991). Masferrer et al. (1990) have speculated that 12(R)-HETE might be an endogenous modulator of Na,K-ATPase in the ciliary epithelium. However, it should be recognized that the HETE family of compounds has a wide range variety of biological effects, many of them concerned with the inflammatory response. For example, both 12(S)-HETE and 12(R)-HETE are recognised neutrophil attractants (Palmer et al., 1980; Wiggins, Jafri and Proia, 19 90). Other arachidonic acid metabolites, such as HHT, also exhibit chemotactic activity for inflammatory cells (Goetzl and Gorman, 1978). In addition, arachidonic acid metabolism itself may be altered by HETES: SHETE appears to be able to stimulate prostaglandin production by cultured MDCK cells (Gordon et al., 1989). The finding that ciliary epitheIium poorly metabolizes arachidonic acid into cyclooxygenase products may be important for the interpretation of prostaglandin effects upon the eye. Prostaglandins are well known for their contribution to inflammatory reactions in the eye (Bito, Nichols and Baroody. 19 82 ; Bhattacherjee, 1989 ; Kulkarni and Srinivasan, 19 89) and certain prostaglandins PGI, (prostacyclin) and PGE, have been shown to cause breakdown of the blood-aqueous barrier (Eakins, 19 7 7 ; Desantis and Sallee, 1989), possibly by altering the permeability of the ciliary epithelial barrier to large molecules (Vegge, Neufeld and Sears, 1975). Furthermore, some cyclooxygenase products are able to lower intraocular pressure (Crawford and Kaufman, 1987; Gabelt and Kaufman, 1989). The low efficiency of prostaglandin production which we observed in ciliary epithelium suggests that prostaglandin-mediated changes in the blood-aqueous barrier at the ciliary epithelium are likely to be caused by prostaglandins produced by tissues outside the epithelium. Acknowledgements This work was supported by USPHS Research Grants .No. EY06915 and EYO6918, the Kentucky Lions Eye Foun-
dation, and an unrestricted grant from Research to Prevent Blindness, Inc. We wish to thank Drs Parimal Bhattacherjee,
ARACHIDONIC
ACID
METABOLISM
BY CILIARY
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Prasad Kulkarni and Christopher A. Paterson for helpful discussion during the course of this work.
References Bazan, H. E. P. (1989). The synthesis and effects of eicosanoids in avascular ocular tissues. In The OcuZurEffects of ProstagZandins and Other Eicosanoids. (Eds Bito, L. 2. and Stjernschantz, J.). Pp. 73-84. Liss: New York. Bhattacherjee. P. (1989). The role of arachidonic metabolite in ocular inflammation. In The Ocular Effects of Prostaglandins andOtherEicosanoids. (EdsBito, L. 2. and Stjernschantz,J.). Pp. 211-27. Liss:New York. Bito, L. Z., Nichols, R. R. and Baroody, R. A. (1982). A comparisonof the miotic and inflammatory effectsof biologically active polypeptidesand prostaglandinE, on the rabbit eye. Exp. Eye Res. 34, 325-37. Blair, I. A. (1990). Electron-capturenegative-ion chemical ionization massspectrometryof lipid mediators.Methods Enzymol. 187, 13-23. Cole, D. F. (1966). Aqueous humor formation. Dot. Ophthalmol.21, 116-238. Crawford, K. and Kaufman, P. L. (1987). Pilocarpine antagonizes prostaglandin F,,-induced ocular hypotensionin monkeys.Arch. Ophthalmol. 105, 1112-6. Davis, K. L., Dunn, M. W. and Schwartzman, M. L. (1990). Hormonal stimulation of 12(R)-HETE, a cytochrome P450 arachidonicacid metabolitein the rabbit cornea. Curr. Eye Res. 9, 661-7. Davson, H. (1990). Aqueous humor and the intraocular pressure.In Physiology of the Eye. (Ed. Davson,H.). Pp. 9-81. AcademicPress:New York. Delamere,N. A., Socci, R. R., Bhattachergee,P. and King K. L. (1991). Studies on the intraocular pressurelowering effect of 12 (R)-Hydroxyeicosatetraenoicacid in the rabbit. Invest. OpthalmoI. Vis. Sci. 32, 25114. Desantis.L. L.. Jr. and Sallee,V. L. (1989). Comparisonof the effectsof prostaglandinsand their esterson bloodaqueousbarrier integrity and intraocular pressurein rabbits.In The Ocular Effects of Prostaglandins and Other Eicosanoids. (EdsBito. L Z. and Stjernschantz,7.). Pp. 379-86. Liss:New York. Eakins,K. E. ( 1977). Prostaglandinand non-prostaglandin mediatedbreakdownof the blood-aqueous barrier. Exp. Eye Res. 25, 483-98.
Escalante, B.. Falck, J. R., Yadagiri, P., Sun, L. and Schwartzman. M. L. (1988). 19(S)-hydroxyeicosatetraenoic acid is a potent stimulator of renal Na+-K+ATPase. Biochem. Biophys. Res. Commun. 152, 1269-74.
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Gabelt,B. T. and Kaufman, P. L. (1989). ProstaglandinF,, increases uveoscleral outflow in the cynomolgus monkey. Exp. EyeRes.49, 389402. Goetzl, E.J. and Gorman, R. R. (1978). Chemotacticand chemokinetic stimulation of human eosinophil and neutrophil polymorphonuclear leukocytes by 12-Lhydroxy- 5,8,10-heptadectrienoic acid (HHT). J. Immunoi. 2, 526-31. Gordon,J. A., Figard, P H., Quinby, G. E. and Spector,A. A. (1989). 5-HETE:uptake, distribution, and metabolism in MDCK cells.Am. J. Physiol. 256, Cl-ClO. Hurst, J. S., Paterson,C. A., Bhattacherjee.P. and Pierce, W. M. (1989). Effectsof ebselenon arachidonatemetabolism by ocular and non-ocular tissues. Biochem. Pharmacol. 38, 3357-63.
Jumblatt, M. M., Raphael,B. and Jumblatt. J. E. (1991). A simplemethod for the isolation of ciliary epithelium. Exp. Eye Res. 52, 229-32.
Kulkarni, P. S., Fleisher,L. and Srinivasan, B. D. (1984). The synthesisof cyclooxygenaseproducts in ocular tissuesof various species.Curr. Eye Res. 3, 447-52. Kulkarni, P. S.andSrinivasan.B. D. (1989). Cyclooxygenase and lipoxygenase pathways in anterior uvea and conjunctiva. In The Ocular Eflectsof Prostaglandins and Other Eicosanoids. (EdsBito. L. Z. and Stjernschantz,J.). Pp. 39-52. Liss:New York. Masferrer, J. L., Dunn, M. W. and Schwartzman, M. L. (1990). 12(R)-hydroxyeicosatetraenoicacid, an endogenous cornea1arachidonate metabolite, lowers intraocular pressurein rabbits. Invest. Ophthnlmol. Vis. Sci. 31, 535-9. Palmer, R. M. J.. Stepney, R. J., Higgs. G. A. and Eakins. K. E. (1980). Chemokineticactivity of arachidonicacid lipoxygenaseproductson ieucocytesof differentspecies. Prostaglandins 20, 411-8. Schwartzman, M. L., Balazy, M., Masferrer. J., Abraham, N. G., McGiff, J. C. and Murphy, R. C. (1987). 12(R)hydroxyeicosatetraenoic acid: a cytochrome P4 50dependentarachidonatemetabolite that inhibits Na’K+-ATPasein the cornea. Proc. Natl. Acad. Sci. U.S.A. 84,
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Vegge, T.. Neufeld, A. H. and Sears, M. L. (19 75). Morphology of the breakdownof the blood-aqueous barrier in the ciliary processesof the rabbit eye after prostaglandin E,. Invest. Ophthnlmol. Vis. Sci. 14. 33-6. Wiggins, R. E., Jafri, M. S. and Proia, A. D. (1990). 12(S)hydroxy-5,8,10,14-eicosatetraenoic acid is a more potent neutrophil chemoattractant than the 12(R) epimerin the rat cornea.Prostuglundins 40, 131-41. Williams,R. N., Delamere,N. A. and Paterson,C. A. (1985). Generationof lipoxygenaseproducts in the avascular tissuesof the eye. Exp. Eye Res. 41, 73 3-8.
