Eq. Eye Res. (1990) 51. 447-450
Effects
of Ultraviolet Irradiation on Prostaglandin-E, by Cultured Cornea1 Stromal Cells ROBERT
Departments
N. WEINREB”,
BEATRICE
Y. J.T.YUE”AND
Production
GHOLAM
A. PEYMANb
of Ophthalmology, University of California, San Diego, La Jolla, CA, “University Chicago, IL, and b Louisiana State University, New Orleans, LA, U.S.A. (Received
17 November
1989 and accepted
in revised
form 8 March
of Illinois,
1990)
We examined the effects of ultraviolet (UV) irradiation on the release of prostaglandin E, (PGE,) by rabbit cornea1 stromal cells in culture. Considerable amounts of PGE, were present in the media of control cornea1 cultures following 1, 2,4, 8 and 24 hr of incubation. Irradiation with UV-A (320400 nm) for 30 min resulted in more than a 50% increase in PGE, release. Dexamethasone inhibited PGE, release by cornea1 stromal cells. It was, however, ineffective in protecting the cells from the UV-induced release of PGE,. Key words: prostaglandin : cornea : ultraviolet : blood-aqueous barrier: ocular inflammation.
1. Introduction
2. Materials
Breakdown of the blood-aqueous barrier can occur following introduction of moderate amounts of prostaglandins into the anterior chamber either by exogenous administration (Camras, Bito and Eakins, 19 77 ; Eakins, 19 77) or through endogenous release from anterior segment tissues after laser photocoagulation of the iris (Weinreb and Mitchell, 1985). A similar response is produced in the rabbit eye upon exposure to ultraviolet (UV) irradiation. The UVinduced ocular changes consist of miosis, conjunctival and iris hyperemia. increased intraocular pressure, and breakdown of the blood-aqueous barrier (Peyman et al., 1986). The exact mechanism of this UV-related action remains unclear. The rabbit cornea absorbs substantial UV radiant energy at short wavelengths (Rmgvold. 1979). For instance, at wavelengths less than 290 nm, the cornea has been shown to absorb greater than 90% of the energy. There is less cornea1 absorption at longer wavelengths. Still, significant energy is absorbed by the cornea at wavelengths less than 400 nm (Sliney and Wolbarsht, 1980; Zigman, 1981). It is possible that the cells in the cornea release prostaglandins into the aqueous humor in response to this UV irradiation. To investigate this possibility, we measured prostaglandins E, (PGE,) release by cultured rabbit cornea1 stromal cells after UV irradiation. Since glucocorticoids are known to inhibit the production of prostaglandins in many biological systems (Gryglewski et al., 1975 : Floman and Zor, 1976 ; Weinreb, Mitchell and Polansky, 1983; Gerritsen et al., 1986), we also investigated the effects of dexamethasone on PGE, biosynthesis in these cells.
TissueCulture Techniques
* For reprintrequests and correspondence. 00144835/90/100447+04
%03.00/O
and Methods
Adult New Zealand albino rabbits were killed and eyes were enucleated. After extensive washing, the middle to posterior portion of cornea1 stroma was isolated and cultured on Coming flasks, as previously described (Yue, Baum and Silbert, 1976). The culture medium included Eagle’s minimum essential medium (MEM), 10% fetal bovine serum, glutamine, essential and non-essential amino acids (all from Hazelton Dutchland, Inc., Denver, PA) and antibiotics. The medium was changed twice a week, unless otherwise stated. When cornea1 stromal cells reached confluency, they were treated with versene and trypsin and subcultured. For the present study, cells in early passages (between passages 3 and 5) were used.
Irradiation Procedure The UV radiation source was a 150 W xenon arc lamp powered by a 150 W power supply (Solar Light Co., Philadelphia, PA). To reduce the transmitted infrared (IR) energy, this radiation source employs a UV-reflecting, IR-transmitting, dichroic mirror in the optic path. The reflected radiation then serially passes through two filters (Schott WG-320 and Coming 9863) before exiting through the housing output port to obtain UV radiation. The output is refocused to obtain a spot 1 cm in diameter at a focal plane 56 mm from the port. To obtain UV-A (320400 nm), a removable filter (Schott WG 345, 2 mm thickness) at the light exit port (Berger, 1969) was added to this radiation source. It should be noted that using the Corning and Schott lilters, the IR and red light irradiation was reduced but not totally eliminated. 0 1990 AcademicPressLimited
R.N.
448
However, the amount of irradiation from these long wavelength regions is insignificant compared with that produced by UV-A. Six days before irradiation, rabbit cornea1 stromal cells were inoculated onto Corning 24-well plates at a density of 80000 cells per well. Four to six wells were set up as controls and another four to six were used as the experimental group. Experiments were performed in parallel with a pair of control and experimental wells. Before each experiment, cells in the experimental and the control wells were washed with serum-free MEM. The cells in the experimental well were then positioned to be exposed to UV-A (320400 nm, 16 mW cmm2) for 30 min. Immediately following irradiation, cells in both experimental and control wells were washed three times and were incubated with O-5 ml of fresh serum-free MEM. The medium was collected and replaced from each well after incubation for 1, 2, 4, 8 and 24 hr. Cells were subsequently harvested and the amount of cell protein was measured by Lowry’s method (Lowry et al., 19 5 1). Experiments were repeated twice. In a parallel experiment, cells were plated and treated with 1 FM of dexamethasone for 22 hr before irradiation to assess the protective ability of this drug against UV irradiation. All the other procedures were the same as described above.
WEINREB
ET AL.
P=oaY: 160
i
140
I
2
II
4
III
6
0 Time
II
IO
12
I4
of incubotlon
I
16
18
III
20
22
FIG. 1. PGE, release from cultured rabbit cornea1 stromal cells exposed (e) and not exposed (0) to UV irradiation. Values represent mean+ S.E.M. (n = 4). P values are determined at each time point. P=o~oml 160T P=O,oO275 /t
140‘-
P
Prostaglandin Assays Radioimmunoassays for PGE, were performed on the media collected from the experimental and the control groups. PGE, was selected because it was shown to be the principal prostanoid biosynthesized by cultured cornea1 stromal cells (Taylor et al., 1982). Sensitive and specific methods for measurements of PGE, were developed previously and validated elsewhere (Mitchell et al., 1982; Weinreb et al., 1983; Weinreb, Weaver and Mitchell, 1985). The lower limit of sensitivity for the assays was approx. 1-5 pg per tube. Cross-reactivities of the antisera with other prostaglandins were less than 1%. Data obtained from four to six duplicate samples were analyzed. Twotailed Student’s t-tests were used to determine the statistical significance of the data. 3. Results
Rabbit cornea1 stromal cells in culture displayed a typically long, thin, fibroblastic morphology. Irradiation with LJV-A for 30 min did not result in any morphological changes in these cells. The effects of the UV irradiation on PGE, release from rabbit cornea1 stromal cultures are shown in Fig. 1. Considerable amounts (12.0, 27.4, 41.8, 59.7 and 71.1 pg ~1 protein-‘) of PGE, were found to be released to the media of control cornea1 cultures following 1, 2, 4, 8 and 24 hr of incubation. Initially, the amount of PGE, present in the media increased linearly, but it
24
(hr)
I 2
I 4
I 6
I 8 Time
I I I IO 12 I4 of incubation
I I6 (hr)
I I8
I 20
I 22
I 24
FIG. 2. PGE, release from cultured dexamethasone-treated rabbit cornea1 stromal cells exposed (0) and not exposed (0) to UV irradiation. Values represent meank~.~.~. (n = 4). P values are determined at each time point. then leveled off after 8 hr. Irradiation with UV for 30 min was associated with at least a 50% increase in PGE, release (33.7, 43.5, 68.3, 139.1 and 158.8 pg pg protein-‘) at the corresponding time points. PGE, release in response to preincubation of the cultured cornea1 stromal cells with dexamethasone is shown in Fig. 2. Dexamethasone inhibited PGE, release and reduced amounts of PGE, were found in the dexamethasone-treated cultures (13.6, 19-7, 28.9, 32.9 and 35-5 pg pg-*) after 1, 2, 4, 8 and 24 hr. At each time point, there was less PGE, release following UV-irradiation from the dexamethasone-treated cultures (27.5, 36.7, 59.0, 115.3 and 144.4 pg ,ug protein-‘) compared with those without the pretreatment, although these differences were not statistically different. 4. Discussion Radiant energy transmitted through the cornea has the potential to damage any intraocular tissue that
PROSTAGLANDIN
PRODUCTION
BY CULTURED
CORNEAL
can absorb it. Aside from direct effects on the lens and retina, it may damage the iris, ciliary body and trabecular meshwork, resulting in changes in aqueous humor dynamics. The longest wavelengths, which have the least energy, are most effectively transmitted through the cornea and interact with intraocular tissues. The cornea absorbs only a small amount of energy of these long wavelengths. However, the shortest wavelengths, which possess the highest energy and are the most damaging to living tissues, are absorbed significantly by the cornea. This is particularly the case for wavelengths less than 320 nm (Sliney and Wolbarsht, 1980; Zigman, 1981). Approximately 50% or more of such radiant energy is absorbed by the cornea and all cells in the cornea should be exposed to a significant fraction of the incident light, even in the presence of a tear layer. In this investigation, we demonstrated that there is substantial PGE, release by the rabbit cornea1 stromal cells following UV-A (320400 nm) irradiation. Corneal stromal cells represent the large majority of cornea1 cells and, hence, might provide a source of PGE, released into aqueous humor. Previously (Peyman et al., 1986) we demonstrated a dose-dependent breakdown in the blood-aqueous barrier in New Zealand albino rabbits with 320400 nm UV-A radiation. Irradiation with 300400 nm produced a significantly greater breakdown in the blood-aqueous barrier than the 320400 nm UV administrated in the same dose (45 J cme2). In fact, animals receiving 300400 nm for 20 min developed cornea1 haziness at 24 hr following termination of exposure, as well as blood-aqueous barrier breakdown 15 min following exposure. It should be noted, however, that despite the present demonstration of prostaglandin release by cornea1 stromal cells, these prostaglandins probably do not contribute to the blood-aqueous barrier breakdown following UV irradiation. Instead, the conjunctiva and the anterior uvea are more likely contributors to the barrier breakdown since prostaglandin biosynthesis in these two tissues is greater than that in the cornea and they may be more affected by the UV irradiation. The UV irradiance of 16 mW crnm2used in this study is approximately 15 times that from the sun in the same wavelength band, 320400 nm, at sea level. It has been shown that the solar radiant power at sea level averages about 0.2 watt per square foot per lonm band between 350 and 400 nm (Carlson and Clark, 1965). For this 50-nm band, the total irradiance is calculated to be approximately 1.1 mW cm-“. Certainly at higher altitudes and at shorter wavelengths, the solar h-radiance is expected to be considerably greater than the l-1 mW crn2 level, While the UV irradiance chosen presently is much higher than the solar irradiance, it is still too low to generate a significant temperature increase in the cultured cell specimens. The dosage of 30 J crne2 used corresponds to about 7 cal cmm2, delivered over a 30-min period.
CELLS
449
Since the cells were not thermally isolated, the temperature rise should be negligible. The culture medium MEM contains riboflavin, tryptophan, and other components that can act as photosensitiirs and do absorb above 300 nm. Prior to the UV exposure, however, the medium was removed from cornea1 stromal cell cultures and during the irradiation, there should not be more than a residual amount of MEIvI present. Immediately following the UV exposure, the cells were washed with fresh MEM and the residual amounts of photosensitized components, if present, were removed. The exposed cells were then incubated with fresh MEM and the production of PGE, with time was monitored. The demonstrated UV-induced effects therefore appear to be unrelated to the photosensitizable components in the culture medium. Our results also indicated that dexamethasone is ineffective in protection of cornea1 stromal cells against UV irradiation. Nevertheless, an inhibition of PGE, secretion following dexamethasone treatment of cells not exposed to UV irradiation was noted. Considerably more investigation is necessary to evaluate the mechanism and to determine whether specific inhibitors of arachidonic acid release are induced by dexamethasone in cornea1 stromal cells. Why dexamethasone is ineffective in protecting the UV-induced prostaglandin release is also unclear at present, One possible explanation is that UV irradiation may have inactivated dexamethasone-induced inhibitors and consequently PGE, biosynthesis would be unchanged. Without UV treatment, these substances are functional, and PGE, biosynthesis is inhibited. Acknowledgments
The authors thank MS Judith Elvart and MS Julie Mackin for their technical assistance. This work was supported in part by N.I.H. grants EY 05990 (RNW), BY 05628 (BY), and EY 03890 (BY). References
Berger, D. S. (1969). Specification and design of solar ultraviolet simulators. 1. Invest. Dermatol. 53, 192-9. Camras, C. B.. Bito, L. 2. and Eakins, K. E. (1977). Reduction of intraocular pressure by prostaglandins applied topically to the eyes of conscious rabbits. fnvest. Ophthalmol. Vis. Sci. 16, 1125-34.
Carlson, F. E. and Clark, C. N. (1965). Light sources for optical devices. In Applied Optics and Optical Engineering (Ed. Kingslake. R.). Vol. 1. Pp. 43-109. Academic Press: New York, NY. Eakins. K. E. (1977). Prostaglandin and nonprostaglandin mediated breakdown of the blood-aqueous barriers. Elcp. Eye Res. 25 (Suppl.), 483-98. Floman, Y. and Zor, U. (1976). Mechanism of steroid action in in8 ammation : Inhibition of prostaglandin synthesis and release. Prostaglandins 12. 403-l 3. Gerritsen, M. E.. Weinstein, B. I.. Gordon, G. G. and Southren. A. L. (1986). Prostaglandin synthesis and release from cultured human trabecular meshwork cells and scleral fibroblasts. Exp. Eye Res. 43, 1089-102.
450
Gryglewski, R. J., Panczenko, G., Korbut, R.. Grodzinska and Ocetkiewicz (19 75). Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig. Prostaglundins 10, 343-55. Lowry, 0. H., Rosebrough.N. J., Farr, A. L. and Randall,R. J. (1951). Protein measurementwith Folin phenol reagent.I. Biol. Chem.193, 265-75. Mitchell, M. D.. Carr, B. R., Mason, J. I. and Simpson,E.R. (1982). Prostaglandinbiosynthesisin the human fetal adrenalgland: Regulationof glucocortico-steroids. Proc. N&l. Acad.Sci. U.S.A. 79, 7547-51. Peyman, G. A., Fishman, P. H., Alexander, K. R., Woodhouse, M. and Weinreb, R. N. (1986). The effect of ultraviolet, visible and infrared radiation on the rabbit blood-aqueousbarrier. Exp. Eye Res. 42. 149-254. Ringvold, A. (19 79). Corneaand ultraviolet radiation. Acta Ophthalmol.61, 898-907. Silney. D. and Wolbarscht, M. (1980). Effect of optical radiation on the eye. In Safety With Lasersand Other OpticalSources (EdsSilney, D. and Wolbarscht,. M.). Pp. 101-60. Plenum Press:New York. Taylor, L., Menconi, M., Leibowitz, H. M. and Polgar. P.
R.N.
WEINREB
ET AL.
(1982). The effectsof ascorbate.hydroperoxides.and bradykinin on prostaglandinproductionby cornea1and lenscells.Invest.Ophthalmol.Vis. Sci. 23, 378-82. Weinreb, R. N. and Mitchell, M. D. (1985). Experimental investigations of intraocular eicosanoids : Cultured human trabecular cellsand laserphotocoagulationof the rabbit iris. Cut-r. Eye Res. 4, 281-90. Weinreb, R. N., Mitchell, M. D. and Polansky,J. R. (1983). Prostaglandinproduction by human trabecular cells: In vitro inhibition by dexamethasone.Invest. OphthaZmoZ. Vis. Sci. 24, 1541-5. Weinreb, R. N., Weaver, D. and Mitchell, M. D. (1985). Prostanoidsin rabbit aqueoushumor: Fffects of laser photocoagulationof the iris. InvestOphthalmol.Vis. Sci. 26, 1087-92. Yue, B. Y. J. T., Baum, J. L. and Silbert, J. E. (1976). The synthesisof glycosaminoglycansby cultures of rabbit cornea1endothelialand stromal cells.Biochem.J. 158, 567-73. Zigman, S. (1981). Photochemicalmechanismsin cataract formation. In Mechanisms of CataractFormationin the Human Lens (Ed. Duncan, G.). Pp. 117-49. Academic Press:New York, NY.
Effects
of Ultraviolet Irradiation on Prostaglandin-E, by Cultured Cornea1 Stromal Cells ROBERT
Departments
N. WEINREB”,
BEATRICE
Y. J.T.YUE”AND
Production
GHOLAM
A. PEYMANb
of Ophthalmology, University of California, San Diego, La Jolla, CA, “University Chicago, IL, and b Louisiana State University, New Orleans, LA, U.S.A. (Received
17 November
1989 and accepted
in revised
form 8 March
of Illinois,
1990)
We examined the effects of ultraviolet (UV) irradiation on the release of prostaglandin E, (PGE,) by rabbit cornea1 stromal cells in culture. Considerable amounts of PGE, were present in the media of control cornea1 cultures following 1, 2,4, 8 and 24 hr of incubation. Irradiation with UV-A (320400 nm) for 30 min resulted in more than a 50% increase in PGE, release. Dexamethasone inhibited PGE, release by cornea1 stromal cells. It was, however, ineffective in protecting the cells from the UV-induced release of PGE,. Key words: prostaglandin : cornea : ultraviolet : blood-aqueous barrier: ocular inflammation.
1. Introduction
2. Materials
Breakdown of the blood-aqueous barrier can occur following introduction of moderate amounts of prostaglandins into the anterior chamber either by exogenous administration (Camras, Bito and Eakins, 19 77 ; Eakins, 19 77) or through endogenous release from anterior segment tissues after laser photocoagulation of the iris (Weinreb and Mitchell, 1985). A similar response is produced in the rabbit eye upon exposure to ultraviolet (UV) irradiation. The UVinduced ocular changes consist of miosis, conjunctival and iris hyperemia. increased intraocular pressure, and breakdown of the blood-aqueous barrier (Peyman et al., 1986). The exact mechanism of this UV-related action remains unclear. The rabbit cornea absorbs substantial UV radiant energy at short wavelengths (Rmgvold. 1979). For instance, at wavelengths less than 290 nm, the cornea has been shown to absorb greater than 90% of the energy. There is less cornea1 absorption at longer wavelengths. Still, significant energy is absorbed by the cornea at wavelengths less than 400 nm (Sliney and Wolbarsht, 1980; Zigman, 1981). It is possible that the cells in the cornea release prostaglandins into the aqueous humor in response to this UV irradiation. To investigate this possibility, we measured prostaglandins E, (PGE,) release by cultured rabbit cornea1 stromal cells after UV irradiation. Since glucocorticoids are known to inhibit the production of prostaglandins in many biological systems (Gryglewski et al., 1975 : Floman and Zor, 1976 ; Weinreb, Mitchell and Polansky, 1983; Gerritsen et al., 1986), we also investigated the effects of dexamethasone on PGE, biosynthesis in these cells.
TissueCulture Techniques
* For reprintrequests and correspondence. 00144835/90/100447+04
%03.00/O
and Methods
Adult New Zealand albino rabbits were killed and eyes were enucleated. After extensive washing, the middle to posterior portion of cornea1 stroma was isolated and cultured on Coming flasks, as previously described (Yue, Baum and Silbert, 1976). The culture medium included Eagle’s minimum essential medium (MEM), 10% fetal bovine serum, glutamine, essential and non-essential amino acids (all from Hazelton Dutchland, Inc., Denver, PA) and antibiotics. The medium was changed twice a week, unless otherwise stated. When cornea1 stromal cells reached confluency, they were treated with versene and trypsin and subcultured. For the present study, cells in early passages (between passages 3 and 5) were used.
Irradiation Procedure The UV radiation source was a 150 W xenon arc lamp powered by a 150 W power supply (Solar Light Co., Philadelphia, PA). To reduce the transmitted infrared (IR) energy, this radiation source employs a UV-reflecting, IR-transmitting, dichroic mirror in the optic path. The reflected radiation then serially passes through two filters (Schott WG-320 and Coming 9863) before exiting through the housing output port to obtain UV radiation. The output is refocused to obtain a spot 1 cm in diameter at a focal plane 56 mm from the port. To obtain UV-A (320400 nm), a removable filter (Schott WG 345, 2 mm thickness) at the light exit port (Berger, 1969) was added to this radiation source. It should be noted that using the Corning and Schott lilters, the IR and red light irradiation was reduced but not totally eliminated. 0 1990 AcademicPressLimited
R.N.
448
However, the amount of irradiation from these long wavelength regions is insignificant compared with that produced by UV-A. Six days before irradiation, rabbit cornea1 stromal cells were inoculated onto Corning 24-well plates at a density of 80000 cells per well. Four to six wells were set up as controls and another four to six were used as the experimental group. Experiments were performed in parallel with a pair of control and experimental wells. Before each experiment, cells in the experimental and the control wells were washed with serum-free MEM. The cells in the experimental well were then positioned to be exposed to UV-A (320400 nm, 16 mW cmm2) for 30 min. Immediately following irradiation, cells in both experimental and control wells were washed three times and were incubated with O-5 ml of fresh serum-free MEM. The medium was collected and replaced from each well after incubation for 1, 2, 4, 8 and 24 hr. Cells were subsequently harvested and the amount of cell protein was measured by Lowry’s method (Lowry et al., 19 5 1). Experiments were repeated twice. In a parallel experiment, cells were plated and treated with 1 FM of dexamethasone for 22 hr before irradiation to assess the protective ability of this drug against UV irradiation. All the other procedures were the same as described above.
WEINREB
ET AL.
P=oaY: 160
i
140
I
2
II
4
III
6
0 Time
II
IO
12
I4
of incubotlon
I
16
18
III
20
22
FIG. 1. PGE, release from cultured rabbit cornea1 stromal cells exposed (e) and not exposed (0) to UV irradiation. Values represent mean+ S.E.M. (n = 4). P values are determined at each time point. P=o~oml 160T P=O,oO275 /t
140‘-
P
Prostaglandin Assays Radioimmunoassays for PGE, were performed on the media collected from the experimental and the control groups. PGE, was selected because it was shown to be the principal prostanoid biosynthesized by cultured cornea1 stromal cells (Taylor et al., 1982). Sensitive and specific methods for measurements of PGE, were developed previously and validated elsewhere (Mitchell et al., 1982; Weinreb et al., 1983; Weinreb, Weaver and Mitchell, 1985). The lower limit of sensitivity for the assays was approx. 1-5 pg per tube. Cross-reactivities of the antisera with other prostaglandins were less than 1%. Data obtained from four to six duplicate samples were analyzed. Twotailed Student’s t-tests were used to determine the statistical significance of the data. 3. Results
Rabbit cornea1 stromal cells in culture displayed a typically long, thin, fibroblastic morphology. Irradiation with LJV-A for 30 min did not result in any morphological changes in these cells. The effects of the UV irradiation on PGE, release from rabbit cornea1 stromal cultures are shown in Fig. 1. Considerable amounts (12.0, 27.4, 41.8, 59.7 and 71.1 pg ~1 protein-‘) of PGE, were found to be released to the media of control cornea1 cultures following 1, 2, 4, 8 and 24 hr of incubation. Initially, the amount of PGE, present in the media increased linearly, but it
24
(hr)
I 2
I 4
I 6
I 8 Time
I I I IO 12 I4 of incubation
I I6 (hr)
I I8
I 20
I 22
I 24
FIG. 2. PGE, release from cultured dexamethasone-treated rabbit cornea1 stromal cells exposed (0) and not exposed (0) to UV irradiation. Values represent meank~.~.~. (n = 4). P values are determined at each time point. then leveled off after 8 hr. Irradiation with UV for 30 min was associated with at least a 50% increase in PGE, release (33.7, 43.5, 68.3, 139.1 and 158.8 pg pg protein-‘) at the corresponding time points. PGE, release in response to preincubation of the cultured cornea1 stromal cells with dexamethasone is shown in Fig. 2. Dexamethasone inhibited PGE, release and reduced amounts of PGE, were found in the dexamethasone-treated cultures (13.6, 19-7, 28.9, 32.9 and 35-5 pg pg-*) after 1, 2, 4, 8 and 24 hr. At each time point, there was less PGE, release following UV-irradiation from the dexamethasone-treated cultures (27.5, 36.7, 59.0, 115.3 and 144.4 pg ,ug protein-‘) compared with those without the pretreatment, although these differences were not statistically different. 4. Discussion Radiant energy transmitted through the cornea has the potential to damage any intraocular tissue that
PROSTAGLANDIN
PRODUCTION
BY CULTURED
CORNEAL
can absorb it. Aside from direct effects on the lens and retina, it may damage the iris, ciliary body and trabecular meshwork, resulting in changes in aqueous humor dynamics. The longest wavelengths, which have the least energy, are most effectively transmitted through the cornea and interact with intraocular tissues. The cornea absorbs only a small amount of energy of these long wavelengths. However, the shortest wavelengths, which possess the highest energy and are the most damaging to living tissues, are absorbed significantly by the cornea. This is particularly the case for wavelengths less than 320 nm (Sliney and Wolbarsht, 1980; Zigman, 1981). Approximately 50% or more of such radiant energy is absorbed by the cornea and all cells in the cornea should be exposed to a significant fraction of the incident light, even in the presence of a tear layer. In this investigation, we demonstrated that there is substantial PGE, release by the rabbit cornea1 stromal cells following UV-A (320400 nm) irradiation. Corneal stromal cells represent the large majority of cornea1 cells and, hence, might provide a source of PGE, released into aqueous humor. Previously (Peyman et al., 1986) we demonstrated a dose-dependent breakdown in the blood-aqueous barrier in New Zealand albino rabbits with 320400 nm UV-A radiation. Irradiation with 300400 nm produced a significantly greater breakdown in the blood-aqueous barrier than the 320400 nm UV administrated in the same dose (45 J cme2). In fact, animals receiving 300400 nm for 20 min developed cornea1 haziness at 24 hr following termination of exposure, as well as blood-aqueous barrier breakdown 15 min following exposure. It should be noted, however, that despite the present demonstration of prostaglandin release by cornea1 stromal cells, these prostaglandins probably do not contribute to the blood-aqueous barrier breakdown following UV irradiation. Instead, the conjunctiva and the anterior uvea are more likely contributors to the barrier breakdown since prostaglandin biosynthesis in these two tissues is greater than that in the cornea and they may be more affected by the UV irradiation. The UV irradiance of 16 mW crnm2used in this study is approximately 15 times that from the sun in the same wavelength band, 320400 nm, at sea level. It has been shown that the solar radiant power at sea level averages about 0.2 watt per square foot per lonm band between 350 and 400 nm (Carlson and Clark, 1965). For this 50-nm band, the total irradiance is calculated to be approximately 1.1 mW cm-“. Certainly at higher altitudes and at shorter wavelengths, the solar h-radiance is expected to be considerably greater than the l-1 mW crn2 level, While the UV irradiance chosen presently is much higher than the solar irradiance, it is still too low to generate a significant temperature increase in the cultured cell specimens. The dosage of 30 J crne2 used corresponds to about 7 cal cmm2, delivered over a 30-min period.
CELLS
449
Since the cells were not thermally isolated, the temperature rise should be negligible. The culture medium MEM contains riboflavin, tryptophan, and other components that can act as photosensitiirs and do absorb above 300 nm. Prior to the UV exposure, however, the medium was removed from cornea1 stromal cell cultures and during the irradiation, there should not be more than a residual amount of MEIvI present. Immediately following the UV exposure, the cells were washed with fresh MEM and the residual amounts of photosensitized components, if present, were removed. The exposed cells were then incubated with fresh MEM and the production of PGE, with time was monitored. The demonstrated UV-induced effects therefore appear to be unrelated to the photosensitizable components in the culture medium. Our results also indicated that dexamethasone is ineffective in protection of cornea1 stromal cells against UV irradiation. Nevertheless, an inhibition of PGE, secretion following dexamethasone treatment of cells not exposed to UV irradiation was noted. Considerably more investigation is necessary to evaluate the mechanism and to determine whether specific inhibitors of arachidonic acid release are induced by dexamethasone in cornea1 stromal cells. Why dexamethasone is ineffective in protecting the UV-induced prostaglandin release is also unclear at present, One possible explanation is that UV irradiation may have inactivated dexamethasone-induced inhibitors and consequently PGE, biosynthesis would be unchanged. Without UV treatment, these substances are functional, and PGE, biosynthesis is inhibited. Acknowledgments
The authors thank MS Judith Elvart and MS Julie Mackin for their technical assistance. This work was supported in part by N.I.H. grants EY 05990 (RNW), BY 05628 (BY), and EY 03890 (BY). References
Berger, D. S. (1969). Specification and design of solar ultraviolet simulators. 1. Invest. Dermatol. 53, 192-9. Camras, C. B.. Bito, L. 2. and Eakins, K. E. (1977). Reduction of intraocular pressure by prostaglandins applied topically to the eyes of conscious rabbits. fnvest. Ophthalmol. Vis. Sci. 16, 1125-34.
Carlson, F. E. and Clark, C. N. (1965). Light sources for optical devices. In Applied Optics and Optical Engineering (Ed. Kingslake. R.). Vol. 1. Pp. 43-109. Academic Press: New York, NY. Eakins. K. E. (1977). Prostaglandin and nonprostaglandin mediated breakdown of the blood-aqueous barriers. Elcp. Eye Res. 25 (Suppl.), 483-98. Floman, Y. and Zor, U. (1976). Mechanism of steroid action in in8 ammation : Inhibition of prostaglandin synthesis and release. Prostaglandins 12. 403-l 3. Gerritsen, M. E.. Weinstein, B. I.. Gordon, G. G. and Southren. A. L. (1986). Prostaglandin synthesis and release from cultured human trabecular meshwork cells and scleral fibroblasts. Exp. Eye Res. 43, 1089-102.
450
Gryglewski, R. J., Panczenko, G., Korbut, R.. Grodzinska and Ocetkiewicz (19 75). Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig. Prostaglundins 10, 343-55. Lowry, 0. H., Rosebrough.N. J., Farr, A. L. and Randall,R. J. (1951). Protein measurementwith Folin phenol reagent.I. Biol. Chem.193, 265-75. Mitchell, M. D.. Carr, B. R., Mason, J. I. and Simpson,E.R. (1982). Prostaglandinbiosynthesisin the human fetal adrenalgland: Regulationof glucocortico-steroids. Proc. N&l. Acad.Sci. U.S.A. 79, 7547-51. Peyman, G. A., Fishman, P. H., Alexander, K. R., Woodhouse, M. and Weinreb, R. N. (1986). The effect of ultraviolet, visible and infrared radiation on the rabbit blood-aqueousbarrier. Exp. Eye Res. 42. 149-254. Ringvold, A. (19 79). Corneaand ultraviolet radiation. Acta Ophthalmol.61, 898-907. Silney. D. and Wolbarscht, M. (1980). Effect of optical radiation on the eye. In Safety With Lasersand Other OpticalSources (EdsSilney, D. and Wolbarscht,. M.). Pp. 101-60. Plenum Press:New York. Taylor, L., Menconi, M., Leibowitz, H. M. and Polgar. P.
R.N.
WEINREB
ET AL.
(1982). The effectsof ascorbate.hydroperoxides.and bradykinin on prostaglandinproductionby cornea1and lenscells.Invest.Ophthalmol.Vis. Sci. 23, 378-82. Weinreb, R. N. and Mitchell, M. D. (1985). Experimental investigations of intraocular eicosanoids : Cultured human trabecular cellsand laserphotocoagulationof the rabbit iris. Cut-r. Eye Res. 4, 281-90. Weinreb, R. N., Mitchell, M. D. and Polansky,J. R. (1983). Prostaglandinproduction by human trabecular cells: In vitro inhibition by dexamethasone.Invest. OphthaZmoZ. Vis. Sci. 24, 1541-5. Weinreb, R. N., Weaver, D. and Mitchell, M. D. (1985). Prostanoidsin rabbit aqueoushumor: Fffects of laser photocoagulationof the iris. InvestOphthalmol.Vis. Sci. 26, 1087-92. Yue, B. Y. J. T., Baum, J. L. and Silbert, J. E. (1976). The synthesisof glycosaminoglycansby cultures of rabbit cornea1endothelialand stromal cells.Biochem.J. 158, 567-73. Zigman, S. (1981). Photochemicalmechanismsin cataract formation. In Mechanisms of CataractFormationin the Human Lens (Ed. Duncan, G.). Pp. 117-49. Academic Press:New York, NY.
