1 Department of Physiology and Biophysics, 3 The Charles B. Stout Neuroscience Mass Spectrometry Laboratory, 4 Department of Neurology, and 5 Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163; and 2 Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, Budapest, Hungary
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ABSTRACT |
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The present study provides evidence that growth factor-like glycerophosphate mediators of the lysophosphatidic acid (LPA) family are present in the aqueous humor and the lacrimal gland fluid of the rabbit eye. By use of a combination of HPLC, two-dimensional TLC, mass spectrometry, and the Xenopus oocyte bioassay, the LPA-like phospholipids LPA, cyclic PA, alkenyl-glycerophosphate (GP), lysophosphatidylserine, and phosphatidic acid were detected as physiological constituents of the fluids bathing the cornea. Corneal injury resulted in an increased production of some of these mediators. Alkenyl-GP, a novel member of the LPA family, has been identified in postinjury aqueous humor, establishing that it is generated endogenously. LPA and its homologues were found to be mitogenic in freshly dissociated keratocytes from uninjured corneas. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury and the increase in LPA-like activity in aqueous humor. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting cellular regeneration of the injured cornea.
lysophosphatidic acid; lipid mediator; chloride current; wound healing; keratocyte; lacrimal gland; aqueous humor
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INTRODUCTION |
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THE CORNEA IS BATHED on its external surface by the tear layer and on its internal surface by the aqueous humor (AH). One component of the tear layer is the protein-rich fluid produced by the lacrimal glands. After corneal injury, it is apparent that growth factors and growth factor-like compounds, generated in lacrimal gland fluid (LGF) and AH, influence the healing process. In the cornea, wound healing involves the proliferation and migration of cells to fill the injured area (41).
In a previous study we demonstrated that serum activates a
Cl current in keratocytes
(ker-ICl)
dissociated from injured but not from uninjured corneas (39). In the
same study we demonstrated that the growth factor-like lipid mediator
lysophosphatidic acid (LPA) mimicked the effects of whole serum,
activating
ker-ICl solely in
keratocytes from injured corneas. Because the concentration of LPA in
serum is sufficiently high to activate
ker-ICl
(3-10 µM) (5), we proposed that LPA-like lipids, at least in
part, were the serum factors responsible for the activation of
ker-ICl. In other
cell types, LPA activates
Cl
currents through a
receptor-mediated pathway (10, 26, 35). Given that LPA is ineffective
in eliciting
ker-ICl from
control uninjured corneas, it would appear that LPA receptors are
either not expressed or not functional in keratocytes isolated from
uninjured corneas (39, 40). On the other hand, hyposmotic stress is also capable of activating
ker-ICl, but,
unlike serum and LPA, it is effective in cells from normal and injured
corneas (39). Activation of
ker-ICl by
hyposmotic stimulus apparently utilizes a receptor or signaling pathway
distinct from that activated by serum and LPA receptors. Nonetheless,
this observation suggests that the
ker-ICl channel
is expressed in uninjured and injured keratocytes and that the
hypotonically activated
ker-ICl may be involved in cell volume control. The responsiveness of LPA after injury
underlines the potentially important role of this group of lipid
mediators in wound healing.
LPA is a lipid mediator abundant in vertebrate serum, where it is generated from activated platelets (5, 8, 36) and other cells (7). LPA elicits a broad spectrum of actions in a variety of cells that range from simple unicellular organisms to mammals (see Ref. 12 for review). In some cell types, LPA stimulates DNA synthesis and cell division (38); in other cells, LPA is antimitogenic (34). LPA has also been shown to prevent programmed cell death in cardiomyocytes and fibroblasts (44). LPA stimulates cell motility and migration (13, 46), which lends further support for its role in the physiological regulation of wound healing.
In Xenopus oocytes, serum with
response thresholds in excess of a
106-fold dilution (33) and
LPA-like lipids in subnanomolar concentrations (20, 35) elicit
oscillatory Cl currents
(oo-ICl). This
LPA-elicited
oo-ICl shows a
highly reproducible dose-response relationship in oocytes from
different frogs, making it a sensitive and simple tool for measuring
LPA-like phospholipids using standard two-electrode voltage-clamp
recording (32, 35). The
oo-ICl is evoked
by LPA-activated G protein-coupled cell surface receptors, stimulating
the phosphoinositide-Ca2+ second
messenger system, resulting in a transient elevation of intracellular
Ca2+. This increase in
intracellular Ca2+, in turn, opens
Ca2+-sensitive
Cl
channels in the oocyte
plasma membrane (33, 35). LPA was identified as the major serum factor
responsible for stimulating this
Cl
current (35).
LPA is only one member of a larger glycerophosphate family found to act
as a mediator of different cellular activities. For example, eight
other lipids that can activate
Cl currents in the oocyte
assay have been detected in serum (35). Although the structure of these
endogenous lipids remains to be elucidated, synthetically prepared
analogs of LPA, including lysophosphatidylserine (LPS) (35) and
1-acyl-sn-glycero-2,3-cyclic phosphate
(cyclic PA) (25), have been shown to activate
oo-ICl. We
recently identified 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate
(alkenyl-GP), in which the hydrocarbon side chain is linked with a
vinyl-ether bond to the glycerol backbone, as a potent agent in
mobilizing intracellular Ca2+
through the activation of distinct subtypes of LPA receptors in the
Xenopus oocyte (29; unpublished
observations). Moreover, using cyclic PA, a receptor subtype-specific
agonist, we demonstrated the simultaneous expression of at least two
different LPA receptors (20). Recently, we cloned one of these
receptors that shows a high affinity and selectivity for LPA (10). In
light of these findings, LPA-like lipids and their receptors are
emerging as a novel lipid mediator-receptor system that may potentially
be involved in the physiological regulation of wound healing and regeneration.
The goal of the present study was to determine whether LPA and/or other LPA-like lipid mediators were present in LGF and AH after corneal injury to the rabbit eye. Our data provide evidence that growth factor-like glycerophosphate mediators are indeed present in AH and LGF. By use of a combination of the Xenopus oocyte bioassay, HPLC, two-dimensional TLC, and mass spectrometry (MS), the growth factor-like phospholipids LPA, cyclic PA, alkenyl-GP, LPS, and phosphatidic acid (PA) were detected as physiological constituents of the biologic fluids bathing the cornea. LPA was also found to be mitogenic in keratocytes freshly dissociated from the uninjured cornea. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury (39) and the increase in LPA-like activity in AH. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting regeneration of the injured cornea.
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MATERIALS AND METHODS |
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Animal rights. Rabbits were treated in accordance with the "Association for Research in Vision and Ophthalmology Statement for Use of Animals in Ophthalmic and Vision Research" as well as the "Guiding Principles for Research Involving Animals and Human Beings" of the American Physiological Society.
Rabbit corneal injury. The procedures have been reviewed and approved by the Animal Care and Use Committee of the University of Tennessee, Memphis. Animals were housed in an American Association for Accreditation of Laboratory Animal Care-accredited facility. Rabbit corneas were injured by transcorneal freezing (40). Briefly, rabbits were put under general anesthesia with xylazine (5 mg/kg) and ketamine (35 mg/kg), and corneas were anesthetized locally with proparacaine (0.5%). Butorphanol tartarate (0.2 mg/kg) was used as a postsurgical analgesic. Corneal freeze wounds were made in both eyes by placement of a liquid nitrogen-cooled brass probe with a 3-mm tip onto the epithelial surface for 40 s. This procedure results in the death of all corneal cells in an ~6- to 7-mm-diameter area, with minimal disruption of the stroma. Rabbits were divided into three groups: control (no wounds), 24-h wound healing, and 90-h wound healing. Each group contained three rabbits, with samples taken separately from the left and right eye.
LGF and AH collection.
LGF was collected by cannulating the lacrimal duct of control and
injured animals (4, 37). After the prescribed wound-healing period,
rabbits were anesthetized as described above. Lacrimal glands were
cannulated, and pilocarpine (200 µg/kg iv) was administered to
stimulate lacrimal secretion. Lacrimal fluid was collected and placed
into preweighed, chilled micropipettes. After lacrimal fluid was
collected from both eyes, rabbits were killed and the anterior chambers
were punctured using a 27-gauge needle. The average volumes of AH and
LGF were 200-300 and 100-200 µl/eye, respectively. Ocular
fluids and lipid extracts were stored under N2 gas at 70°C.
Electrophysiological recording from Xenopus oocytes.
Oocytes were obtained from xylazine-anesthetized adult frogs
(Xenopus laevis; Carolina Scientific,
Burlington, NC) under aseptic conditions and prepared for the
experiments as described previously (33). Briefly, electrophysiological
recording was carried out during the first 5 days after defolliculation
of the oocytes with collagenase. Oocytes were voltage clamped at
60 mV with a standard two-electrode voltage-clamp amplifier
(GeneClamp 500, Axon Instruments, Foster City, CA) during superfusion
with frog Na+-Ringer solution (in
mM: 120 NaCl, 2 KCl, 1.8 CaCl2, 5 HEPES; pH 7.0) at a flow rate of 5 ml/min. Membrane currents were
monitored with a digital oscilloscope (model NIC-310, Nicolet, Madison, WI).
Chemicals.
Oleoyl-LPA, dioleoyl-PA, and oleoyl-LPS were purchased from Avanti
Polar Lipids (Alabaster, AL) and stored under
N2 at 20°C. Palmitoyl
cyclic LPA was synthesized as described previously (19). HPLC-grade
solvents were purchased from J. T. Baker Chemical (Phillipsburg, NJ)
and from Fisher Scientific (Pittsburgh, PA). All other chemicals were
purchased from Sigma Chemical (St. Louis, MO) unless otherwise specified.
HPLC. Lipid extracts were chromatographed on a 5-µm silica-packed semipreparative Microsorb Si column (250 × 10 mm; Rainin Instruments, Woburn, MA). A Waters HPLC system controlled by the Millennium 2010 software package (version 2.1, Waters Instruments, Milford, MA) was used for solvent delivery and injection. Elution of the lipids was monitored by an evaporative light-scattering detector (model IIA, Varex, Burtonsville, MD) through a metering valve, splitting the effluent 1:4 between the detector and a fraction collector, respectively.
A modification of the gradient elution originally described by Bünemann et al. (2) was used. Solvent A was made up of 60:35:4.5:0.5 (vol/vol/vol/vol) chloroform-methanol-water-28% ammonium hydroxide; solvent B consisted of the same compounds with relative proportions of 30:60:9.5:0.5, respectively. Solvent A was applied for 10 min, and a gradient of the two solvents was started by increasing solvent B to 100% over a period of 30 min followed by a 40-min isocratic elution at a flow rate of 2 ml/min throughout the run.TLC. K6 silica plates, 20 × 20 cm with 250-µm layer thickness, were used (Whatman, Fairfield, NJ). Two-dimensional TLC separations were done using solvents of 6:4:1 (vol/vol/vol) chloroform-methanol-28% ammonium hydroxide in the first dimension and 6:8:2:2:1 (vol/vol/vol/vol/vol) chloroform-acetone-methanol-glacial acetic acid-water in the second dimension (36). Lipid spots were visualized after primulin (Aldrich Chemical, Milwaukee, WI) staining (43). Lipid spots marked with a pencil were scraped off the plate into glass vials, and lipids were eluted from the silica gel with methanol. Extraction was repeated three times for 5 min each, followed by centrifugation. The supernatants were filtered through a 0.2-µm Teflon syringe-filter and dried under nitrogen.
MS. Mass spectra were obtained by using the fast atom bombardment (FAB) technique in the positive and negative mode on a tandem mass spectrometer [AutoSpecQ E1BE2qQ, VG Fisons (now Micromass), Altrincham, UK] and the VG Opus level 1.7f software package for data analysis. A glycerol matrix (1 µl) was applied, and CsI was used for mass calibration.
Assay of LPA-induced DNA synthesis. Keratocytes were dissociated from uninjured corneas taken from rabbits anesthetized as described above. Corneal endothelium and Descemet's membrane were completely peeled off the stroma with a jeweler's forceps. The remaining epithelium and stroma were incubated in 1.2 U/ml dispase (grade II, Boehringer Mannheim, Indianapolis, IN) in MEM for 1 h at 37°C. The epithelium was peeled off, and the stroma was cut into 1- to 2-mm blocks and incubated with 1 mg/ml collagenase (type IA, Sigma Chemical) in MEM for an additional 2 h at 37°C. Cells were triturated with a fire-polished glass pipette and centrifuged at 180 g for 7 min. Freshly dissociated corneal cells were plated on 24-well plates (Sarstedt, Newton, NC) at a density of 5 × 104 cells/well in a 50:50 DMEM/F-12 medium (Cellgrow, Mediatech, Pittsburgh, PA). Wells, in triplicate, received solvent (30 µM BSA in Hanks' buffered saline solution), 10% FCS, or 10 and 30 µM LPA (18:1), alkenyl-GP (18:1), and LPS (18:1), all complexed with 30 µM BSA. DNA synthesis was determined by measuring the incorporation of [methyl-3H]thymidine (Amersham Life Sciences, Arlington Heights, IL; sp act 185 GBq/mmol). After 21 h in the presence of the mitogens, 1 µCi [methyl-3H]thymidine was added per well, and cells were harvested 3 h later using a cell harvester (Brandel, Gaithersburg, MD). The incorporated acid-insoluble radioactivity was determined by liquid scintillation counting using a Beckman LS 5000TA counter.
Statistical evaluation. Values are means ± SE. Student's t-test was used to determine whether injury-induced changes in the LPA-like activity were statistically significant. P < 0.05 was considered significant.
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RESULTS |
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LGF and AH activate oo-ICl.
LGF and AH were collected from uninjured eyes (control) as well as from
eyes with injured corneas 24 and 90 h after injury. Aliquots were taken
from the eyes separately; the collected fluids were rapidly frozen and
stored at 80°C until further processing. LGF and AH sample
aliquots were tested in the oocyte bioassay for LPA-like activity at
100-fold dilution in frog Ringer solution by a blinded investigator in
a random sequence. The dose-response relationship for the LPA-evoked
oo-ICl was
calibrated for every oocyte, with oleoyl-LPA as a standard. The
responses elicited by LGF and AH samples were determined for three
oocytes and normalized to the mean response of 92 ± 11 nA
(n = 15 oocytes from 5 frogs) elicited
by 1 nM oleoyl-LPA, which was arbitrarily chosen to be 1 response unit
(1 U = mean response to 1 nM oleoyl-LPA = 92 nA). Figure
1 shows the means of the
normalized oo-ICl
responses elicited by LGF and AH samples collected 24 and 90 h after
injury compared with the specimens collected from the uninjured eye.
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Lipid content of LGF and AH. To determine the active component(s) in AH and LGF, polar lipids were extracted with methanol. For control, 4.1 g of AH and 2.4 g of LGF (wet weight) were collected from eyes of rabbits (n = 12) with normal, uninjured corneas. These fluids were lyophilized before extraction with methanol. Because of the limited amount of AH and LGF, samples collected at 24 and 90 h after injury were pooled; the total yield of 0.48 g (wet weight) of AH and 0.84 g of LGF liquids was lyophilized. The freeze-dried specimens were weighed, and lipids were extracted three times with vigorous shaking for 10 min in 5 ml/g (dry wt) methanol containing 50 µg/ml butylated hydroxytoluene as antioxidant. After centrifugation for 10 min at 2,000 g, supernatants were collected, combined, dried under a stream of N2 at 40°C, and weighed.
Data obtained for the dry material and polar lipid content of AH and LGF are summarized in Table 1. In control corneas the dry-to-wet weight ratio was 66 mg to 4,100 mg (1.6%) for AH and 98 mg to 2,400 mg (4.1%) for LGF. For injured eyes, these values were 8.4 mg to 480 mg (1.8%) and 42 mg to 840 mg (5.0%), respectively. The proportion of methanol-soluble lipids showed a substantial difference between AH and LGF. A total of 55 mg of the 66 mg of dry material (83%) was methanol-soluble lipid in control AH. In contrast, only 20 mg of the 98 mg of dry material (20%) were methanol-soluble lipid in control LGF. In samples taken from injured eyes, 7.4 mg of the 8.4 mg of dry material (88%) and 6.9 mg of the 42 mg of dry material (16%) were methanol-soluble lipid in AH in LGF, respectively.
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Fractionation of the methanol-soluble lipids by HPLC.
To further characterize the polar lipids extracted by methanol, an HPLC
method was developed for the separation of the major phospholipid
classes. Lipids were dissolved in solvent
A at a concentration of 10 mg/ml, and a total of 20 mg
was injected into the column. Representative chromatograms of the
extracts from normal corneas are shown in Fig.
2 for AH and LGF. Five-minute fractions
were collected, dried under N2,
and redissolved in 500 µl of methanol. LPA-like activity present in
the fractions was tested using the oocyte bioassay. LPA-like activity
was detected in the fractions that eluted from 5 to 15 min at a
100-fold dilution and in the region that eluted from 25 to 45 min at a
1,000-fold dilution (Fig. 2, bars). The active fractions of AH and LGF
in the two regions were pooled separately and dried under a stream of
N2. The active fractions are
referred to as AH-(515), AH-(25
45), LGF-(5
15), and LGF-(25
45).
The retention times for different known phospholipid standards under
the conditions of this experiment are given in Table
2. The first peak of activity showed a
retention time similar to that of cyclic PA (7 min), whereas the second peak of activity overlapped with the retention times of LPA,
alkenyl-GP, PA, and LPS (Table 2).
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Two-dimensional TLC analysis of AH and LGF lipids.
To identify the active phospholipid components in LGF and AH,
two-dimensional TLC separations were carried out with the active HPLC
fractions LGF-(2545) and AH-(25
45) obtained from control corneas and the unseparated methanol extract of AH from injured corneas. Samples were reconstituted in a mixture of 1:2 (vol/vol) chloroform-methanol, and ~200 µg of lipid were spotted per plate. The plates were developed first in the basic and then in the acidic solvent system, stained with primulin, and viewed under ultraviolet illumination. Stained spots of lipids were marked with a pencil, scraped from the plate, and eluted with a large excess of methanol. Figure 3 illustrates representative TLC
patterns, showing the lipid species present in LGF-(25
45),
AH-(25
45), and AH from injured corneas. Individual lipid spots eluted
from the TLC plate were reconstituted in 200 µl of methanol and
tested for activation of
oo-ICl using the
bioassay.
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FAB-MS spectra of the active TLC spots.
After activity tests using the oocyte bioassay, the active components
were concentrated to a volume of ~20 µl under a stream of
N2 and subjected to MS analysis
using FAB-MS in the positive and negative mode. MS analysis of the
active spots corroborated, via the
(m-H)
ion, the identity of these naturally occurring molecules with those of
the comigrating TLC standards; those data reveal the presence of
components with molecular masses corresponding to LPA, PA, and LPS,
with different hydrocarbon chains (data not shown). LPA, PA, and LPS
have long been known as physiological constituents of biologic fluids
(8, 36). In contrast, alkenyl-GP has not previously been identified as
an endogenous constituent of a physiological fluid. The negative-ion
FAB-MS spectra of the lipid recovered from spot
6 and that of an alkenyl-GP standard are shown in Fig.
5, A and
B, respectively. Because of its
relative abundance, the high signal-to-noise ratio that was obtained
allowed the identification of the major molecular species present in
this spot. The compound with an m/z
of 421 corresponds to an 18:0 aliphatic chain-substituted alkenyl-GP
(m422/z-H+ = 421). In
addition, C18-1 aliphatic chain-substituted alkenyl-GP was
detectable with an m/z of 419.
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LPA-induced DNA synthesis in freshly dissociated keratocyte cultures. Cultures of freshly dissociated corneal keratocytes from uninjured rabbits (n = 3) were treated with 10 and 30 µM LPA, alkenyl-GP, or LPS immediately after isolation or with 10% FCS to establish whether these lipids induce cell proliferation in corneal keratocytes (Fig. 6). All three lipids, at both concentrations, caused a significant increase in [3H]thymidine incorporation. Therefore, it appears that corneal cells acquire mitogenic responsiveness to LPA-like lipids within the first 24 h of their isolation.
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DISCUSSION |
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Keratocytes residing within the collagen network of the corneal stroma
play an essential role in repair after injury. Keratocytes normally are
quiescent but, when activated, migrate to the site of injury and
proliferate, constituting the wound-healing process. Activated
keratocytes generate proteases (15, 16); phagocytose foreign bodies and
injured tissue (22); produce a variety of mediators, including
interferon, prostaglandin, and complement factor I (23, 30, 31);
secrete components of the extracellular matrix, including fibronectin,
collagen, and proteoglycans (1, 18); and show an increase in thymidine
incorporation, indicating their proliferation (42). Epithelial growth
factor (28), fibroblast growth factor (11), interleukin-1,
transforming growth factor- (9), insulin (42), retinoic acid, and
serum (14) have been shown to cause activation of keratocytes. Serum
initially captured our attention as a keratocyte activator because of
the abundance of compounds present in serum that have been found to
enhance growth. A major group of growth-promoting agents in serum,
however, does not belong to the polypeptide growth factor family but
are phospholipids. Before this report, there were no reports available concerning the presence of growth factor-like phospholipids in the
biologic fluids bathing the cornea.
LPA is the best-characterized member of a family of lipid mediators capable of positive or negative regulation of cell proliferation (35, 34). We and others have shown that, along with LPA, alkyl-GP, cyclic PA, LPS, and PA can activate LPA receptors and elicit LPA-like responses (20). We have also demonstrated the expression of at least two distinct receptors for glycerophosphatidates, which are pharmacologically distinguished through cyclic PA responsiveness. The subtype-specific LPA receptor agonist cyclic PA has been shown to be a stable intermediate produced by phospholipase D (6), an enzyme that is under tight regulation by hormones and growth factors. After our initial report that LPS also elicited LPA-like effects in Xenopus oocytes and PC-12 cells, Xu and co-workers (45) demonstrated a complete heterologous desensitization between LPA and LPS, indicating that LPS acts through LPA receptors. More recently, we isolated and synthesized a novel LPA analog, alkenyl-GP (29), that until now had not been shown to be a naturally occurring component of any biologic fluid.
LPA has been shown to activate a
Cl conductance in
keratocytes from injured, but not from control, corneas (39).
Twenty-four hours after injury was sufficient for the occurrence of LPA
responsiveness, which was maintained for at least 4 days. Here, we
applied the Xenopus oocyte bioassay to
detect and monitor the changes in LPA-like activity of LGF and AH
samples collected at 24 and 90 h after injury and in control eyes with
uninjured corneas. LGF and AH showed characteristic changes during
wound healing (Fig. 1). The LPA-like activity in LGF was low and did
not change significantly after 24 and 90 h of healing. In contrast, a
significantly higher basal level of LPA-like activity was found in
control AH, and this activity increased rapidly, by as much as
~13-fold, 24 h after injury and remained high for up to 90 h. It is
important to point out the corresponding occurrence of LPA
responsiveness in keratocytes and the increase in the LPA-like activity
in AH. These data indicate that the LPA mediator family and its
receptors could play a physiological role in corneal wound healing.
This hypothesis is supported by the inducible nature of some of the recently identified putative LPA receptor genes (21). Further experiments are necessary to establish the expression of the different LPA receptor subtypes and their regulation in resting and activated keratocytes.
The role of lipid mediators in the physiological maintenance of corneal integrity and regeneration is underlined by the unexpectedly high methanol-soluble phospholipid content of AH (Table 1). Despite the similar overall lipid content of AH (1.4%) and LGF (0.8%), the dry materials of AH consisted of ~90% polar lipids in contrast to the 20% lipid content of LGF. The high lipid content of AH suggests that it is almost like a lipid suspension, whereas LGF is a solution with a smaller, but still significant, lipid content. In the context of the relatively high lipid content of these ocular fluids, the lipid-soluble LPA-like mediators could play a role in the normal cellular function of the cornea, where there are intact cellular barriers, as well as in injured corneas, where the epithelial and endothelial cell barriers are disrupted. Moreover, given that AH and LGF bathe ocular tissues other than the cornea, including the iris, lens, ciliary body, conjunctiva, and sclera, it is possible that these lipid mediators play a physiological role throughout the anterior portion of the eye.
HPLC fractionation of the methanol-extractable lipids of LGF and AH
from normal eyes showed two major fractions with LPA-like active lipids
(Fig. 2). The first active peak eluted with a retention time in good
agreement with that of cyclic PA (7 min). Consequently, cyclic PA is
the most likely LPA-like compound present in the first active peak in
LGF-(515) and AH-(5
15). The second active broad peak eluted near
the LPA standard, between 25 and 45 min. The retention time of this
second group of activities overlaps that of LPA analogs, including LPA,
alkyl- and alkenyl-GPs, and LPS. Two-dimensional TLC and MS have
corroborated the presence of different members of the LPA family in LGF
and AH (Fig. 3, Table 3). LGF and AH from control corneas contain PA
and LPA. Using negative-ion FAB-MS, we have, for the first time, been
able to positively identify the presence of alkenyl-GP molecules in AH
from injured corneas, establishing that this LPA analog also occurs
endogenously. The level of LPS in LGF, if any, was below the limit of
detectability of the assay systems used in the present study. In
contrast, we found LPS to be abundant in AH from control corneas. LPS
has a very high (20 µM) EC50 in
oocytes (unpublished observations), whereas LPA, alkenyl-GP, and
alkyl-GP have EC50 values in the
low nanomolar range. This difference places LPS as a low-potency
agonist in the LPA-like mediator family, which can potentially
synergize with other mitogens. The relatively high activity and
apparent abundance of spot 5 in
AH-(25
45), which comigrates with LPS, suggest the presence
of a high amount of LPS in the AH in the normal eye (Table 4). In
addition, we have detected a few additional active minor compounds in
LGF and AH, the identities of which are yet to be determined (Table 3). The observation that LPA and LPS were not among the detectable active
components in injured AH is an interesting one. We do not know whether
there is a true decrease in the relative abundance of these two lipids
while other AH components, like alkenyl-GP, increase after injury.
Nonetheless, more research is necessary to establish the biosynthetic
and breakdown pathways for each of these lipids in the blood-free
environment of the ocular anterior chamber.
We have also provided evidence that LPA-like lipids, after a 24-h
incubation, induce keratocyte proliferation, as revealed by increased
thymidine incorporation into freshly dissociated keratocytes from
uninjured eyes. This observation, together with the lack of
LPA-elicited Cl currents in
freshly dissociated keratocytes from uninjured eyes (39), might suggest
a rapid induction of LPA responsiveness within the first 24 h after
cell dissociation and placement into culture. One possible explanation
for the development of LPA responsiveness would be de novo LPA receptor
expression, given that the signaling pathway leading to
Cl
channel opening could be
activated by an osmotic stimulus in freshly isolated keratocytes that
were unresponsive to LPA stimulation (39). The induced expression of
LPA receptor subtypes during keratocyte activation is under
investigation in our laboratories.
The present study establishes that growth factor-like glycerophosphate mediators are present and are generated in increased amounts after injury in AH and LGF. Furthermore, we provide evidence that analogs of the mitogenic lipid mediator LPA, including cyclic PA, LPA, LPS, PA, and, for the first time in any biologic system, alkenyl-GP, are physiological constituents of the biologic fluids bathing the cornea. We also show that LPA is mitogenic in freshly dissociated cells from the uninjured cornea, suggesting that LPA and its homologues are involved in maintaining the integrity of the control cornea and in promoting the regeneration of the injured cornea.
In summary, we propose that the novel group of LPA-like mediators that are abundant in the lipid-rich fluids bathing the cornea can potentially be important physiological regulators of ocular integrity and regeneration.
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ACKNOWLEDGEMENTS |
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We thank Dr. John Ubels for demonstrating the LGF collection technique.
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FOOTNOTES |
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This work was supported by National Science Foundation Grant IBN-9321940 (G. Tigyi) and National Eye Institute Grant EY-10178 (M. A. Watsky). G. Tigyi is an Established Investigator of the American Heart Association.
Address for reprint requests: G. Tigyi, Dept. of Physiology and Biophysics, College of Medicine, University of Tennessee, 894 Union Ave., Memphis, TN 38163.
Received 16 September 1997; accepted in final form 16 December 1997.
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