Cultured Bovine Corneal Epithelial Cells Express a

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IOVS, January 1999, Vol. 40, No. 1 5. Hutchinson IV, Alam Y, AylilTc WR. The humoral response to an allograft. Eye. 1995:9:155-160. 6. Treseler PA, Santilippo F. Humoral immunity to heterotopic corneal allografts in the rat. Transplantation. 1985:39:193-196. 7. Hedge S, Hargrave S, Mellon J, Nieclerkorn JY. Antibody- and cell-mediated immune responses during rejection of" a murine orthotopic conical graft [ARVO Abstract |. Invest Opbtbalmol Vis Sci. 1998;39(4):S455. 8. llahn All. Foulks GN. linger C. et al. The association of lymphocytotoxic antibodies with corneal allograft rejection in high risk patients. The Collaborative Corneal Transplantation Studies Research Group. Transplantation. 1995:59:21-27. 9. Kitamura D, Roes J. Kiihn R, Rajewsky K. A U cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin ii chain gene. Mature. 199l;35O:423-426.

10. Binning J\V, Claas FHJ, Karclol MJ, Lansbcrgcn Q, Naipal AM, Tankc 11). Automated reading of HI.A-A.H,C typing and screening: the propidiuni iodide (PI) method. Hum Immunol. 1982;5:225-231. 11. Wessels MR. Butko P, Ma M, et al. Studies of group 11 streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity. Proc Natl Acad Sci. 1995:92: 1 1490-11494. 12. Sonoda Y. Streilein JW. Orthotopic corneal transplantation in mice: evidence that the immunogenetic rules of rejection do not apply. Transplantation. 1992;54:694-7()4. 13- He YG, Ross J, Nicderkorn JY. Promotion of" murine orthotopic corneal allograft survival by systemic administration of anti-OM monoclonal antibody. Invest Opbtbalmol Vis Sci. I99I;32:27232728.

Cultured Bovine Corneal Epithelial Cells Express a Functional Aquaporin Water Channel

inhibited by 56% to 58% by coinjection with aquaporin (AQP)5 antisense oligonucleotide. CONCLUSIONS. The comparatively high Pf determined lor CBCEPCs, the presence of mRNA encoding water channels, and sensitivity to mercurial agents are typical of the expression of functional water channels. The predominant message is for AQP5, although the evidence was consistent with the presence of additional water channels. These findings bring renewed support for the notion that the epithelium can contribute to corneal hydration homeostasis. (Invest Ophthalmol Vis Sci. 1999;40: '253-257)

Fengying Kang,' Kunyan Kuang,' Jun Li,' and Jorge Vischbarg'l2 Puwosn. Given recent physiological and in situ hybridization evidence for the presence of" a water channel in corneal epithelium, this study was conducted to investigate its expression and characteristics using cultured bovine corneal epithelial cells (CBCEPCs). METHODS. CBCEPCS were grown in DMEM containing 2

ng/ml fibroblast growth factor and 6% fetal bovine serum. To determine their osmotic permeability (A*,), cells were passaged onto rectangular glass coverslips, and anisotonically induced volume changes were monitored by light scattering. To investigate expression, poly(A + ) RNA from CBCEPCs was injected into Xenofnis laevis oocytes, and the /^ of the oocytes was determined. RESULTS. For CBCEPCs challenged with a 10% hypotonic solution at 37°C, the kinetic constant of volume change was k = 0.52 ± 0.04 seconds" 1 , and the calculated Pf 72 ± 6 /xm/sec (ri = 16). The /^of oocytes injected with water was 14 ± 1.8 /xm/sec (// = 4); injection with poly(A") RNA from CUCEPCs increased Pf to 77 ± 6 /urn/sec (« = 6). This increase in Pf was inhibited by 72% (reduced to 22 ± I /xm/sec) by 0.3 m\l HgCI2 and was

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lthough the barrier function of the stratified corneal epithelium is well established, the epithelial role in the regulation of stromal hydration has remained controversial. Still, this epithelium has been recognized to have high osmotic permeability.1 In addition, corneal epithelial transport has been linked to corneal hydration homeostasis, 2 and isolated reports have appeared showing evidence of some fluid transport by corneal epithelium in rabbit^ and frog'1 preparations. The subject gained in clarity more recently when it became known that chloride activates water permeability in the frog corneal epithelium, 5 the mRNA of frog corneal epithelium encodes a water channel, 6 and the water channel AQP5 was located in rat corneal epithelium by hybridization immunohistochemistry.' We have investigated this matter further using cultured bovine corneal epithelial cells (CBCEPCs). We have confirmed the presence of AQP5 in corneal epithelium and have determined that it is functional and that its message seems to be the predominant one for these cells.

A

MATERIALS AND METHODS I'rom the Departments of 'Ophthalmology and Physiology and ''Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York. Supported by Grant LY06178 ()l:) from the National Institutes of Health, Methesda. Maryland; and Research to Prevent Blindness. New York, New York. Submitted for publication Icbruary 27, 1998; revised July 17, 1998: accepted September 8, 1998. Proprietary interest category: N. Reprint requests: Jorge l:ischbarg, Department of Ophthalmology, College of Physicians anil Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032.

Cultured Bovine Corneal Epithelial Cells The primary culture methods used were established in Cubitt et al." Briefly, bovine corneas were surgically isolated, and the endothelium along with part of the stroma was removed by peeling. The resultant preparation was incubated with neutral protease (Dispase II; Gibco, Grand Island, NY) at 25 units/ml at 37°C for 40 minutes to separate the epithelium from its basement membrane, after which Dulbecco's modified Eagle's medium containing 6% fetal bovine serum (Atlanta Biologies,

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fit, k=0.79 s"1, P,=109 urns'1 fit, k=0.48 s"1, P(=65 urns'1

pCMBSj.-.—-- fit, k=0.16 s'1, P=22 (jms"1

1.081.061.041.02-

O

1.000

10

20

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Time (s) FIGURE I. Effect of/>-chloromerciiribenzenesulfonatc (pCMBS) on Pf of cultured bovine conical epithelial cells (CBCIil'Cs). During given experiments, three consecutive osmotic challenges with a 10% hypotonic solution were imposed on the same set of plated cells; a sample experiment is shown. ( • ) Control challenge. (O) Challenge after 20-minule preincubalion with 1 m.M /;CM13S. (A) Challenge after pCMBS was removed by washing with isotonic solution containing 5 mM dithiothreitol for 5 minutes. Curves were lit by the computer program Amoeba.9 written in Power Basic. Inset: average !'/•(— SI-) values for plated CBCEPCs. Numbers within bars are the number of CBCEPC-coniaining glass covcrslips in each group of experiments.

Norcross, GA) was added to neutralize the enzyme. The loosened epithelial cells were scraped off and were further dispersed by passing them through a 22-gauge needle. After they were washed twice by resuspension and centrifugation, the cells were seeded into flasks (Falcon Labware, Oxnard. CA) and were grown in Dulbecco's modified Hagle's medium containing 6% fetal bovine serum, 2 ng/ml basic fibroblast growth factor, 100 U/ml penicillin, and 100 ju-g/ml streptomycin and were maintained at 37°C in a 5% CO 2 incubator. The medium was changed twice weekly. After 1 week, the cells reached confluence.

Measurements of Osmotic Water Permeability in CBCEPCs Cells were used for l3t measurements up to the third passage. For these measurements, we determined the intensity of light scattered by plated CBCEPCs on exposure to a 10% hypotonic solution. The values of the kinetic constants for the osmotic transients (k), unstirred layer thickness (5), and the resultant Pf. were determined as previously described. 91 " For this, cells were passaged onto sterile rectangular glass coverslips (11 X 22 mm; Thomas Scientific. Swedesboro, NJ) and were cultured for an additional 36 to 48 hours. The optimum cell density on the coverslips was approximately 80% to 90% of confluence. For this study, the perfusion solution contained (in millimolar) 107.9 NaCl, 26.2 NaHCO3, 4.74 KCI, 1.0 NaH2PO/f, 0.39 MgSO4 (7 H2O), 1.8 CaCI2 (2 H2O), 5 5 glucose, and 2 2 9 HEPES-Na (300 mOsM). For osmotic challenge, we obtained a 10% hypotonic solution (270 mOs.M) by reducing the |NaCI|. The osmo-

larity of the solutions used was confirmed using a /u-Osmette instrument (Precision Systems, Natick, MA). In some experiments, the perfusion solutions contained />chloromercuribenzenesulfonate (pCMUS), mercuric chloride (HgCI2), dithiothreitol, or /3-mercaptoethanol (all from Sigma, St. Louis, MO). Solutions were prepared just before the experiments.

Preparation of RNA Total RNA from passages one through three of CBCEPCs was isolated by a commercial method (RNAzol; Biotecx Laboratories, Houston. TX). PolyOV") UNA was purified by affinity chromatography on oligo deoxythymicline cellulose columns. Two mouse AQPI oligonucleotide primers were used as described previously.1" In addition, we used two rat AQP5 oligonucleotide primers: antisense, +16 to +40 (5'-CCGCCTTTGAAGAAGGCAAGGGAGCA-3'); and sense, +14 to +38 (5'TGTGCrCCC'nGCCITCnCAAGGC-3'). These primers were synthesized by Oligos Etc. (Wilsonville, OK). For in vitro mRNA transcription, rat AQPI cRNA was prepared as previously described. 1 "

Preparation and Injection of Oocytes and Determination of Pf Oocytes were prepared as described previously10 from mature female Xenopus laevis frogs. Treatment of animals was in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Oocytes were injected using a microinjector (Picospritzer II; General Valve, Fairfield, NJ) microinjector. Injections were 50 nl water for control, 50 ng

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CBCEPC mRNA

AQP1 cRNA

oocytes injected with: FIC'IUKI-: 2. Expression of water cliannels in batch 1. Oocyies were injected with water, cultured bovine conical epithelial cell (CHCF.I'C) poly(A" ) UNA. or aquaporin (AQP)l cRNA, and the effects of AQPl or AQ1J5 sense or antisense oligoniicleotides and Hj;CI2 on the expressed Pf were determined. Bars, mean ± SI:; numbers within bars are the number of oocytes in each group.

poly(A ' ) RNA of CBCEPCs or 0.2 ng AQPl cRNA in 50 nl water, AQPl or AQP5 sense or antisense oligoiiucleotides in 50 nl water (2.5 ng/oocyte), or coinjected with 50 ng of poly(A+) UNA of CBCIiPCs or 0.2 ng AQPl cRNA in 50 nl water. We assumed that the fraction of poly(A') RNA encoding water channels represented only 1% of the total poly(A+) RNA. and therefore injecting the amount of antisense oligoniicleoticle noted would exceed its target mRNA by perhaps 100 times on a molar basis. The oocytes were assayed 4 clays later; Pf was determined from the time course of the change in oocyte volume when exposed to a hypotonic solution.10 For the /^determinations, oocytes were placed in a glass-bottomed chamber (—0.3 ml capacity) containing Bartlrs buffer at room temperature. The oocyte was viewed with an inverted microscope (T.V1S; Nikon. Garden City, NY) equipped with a video camera (model NC-6; Dagc-MTl, Michigan City, IN) connected to a monitor screen. The oocyie was superfused first in 178 mOsM isotonic Barth's buffer for 60 seconds and then in hypotonic 15 mOsM Barth's buffer for 90 seconds while an image frame of the oocyte was recorded every 10 seconds with a video digitizer board. The Pf was calculated from the initial change in oocyte volume in response to the hypotonic solution: Pf = (c/V/ df) X I /(A X Vw X AQ, where A is the total (spherical) area of the oocyte at zero time. clV/clt is the initial slope of the oocyte volume versus time, Vw is the molar volume of water, and AC = (0.178 - 0.015) M is the osmolarity gradient at zero time (assuming 0.178 M is isotonic to the intracellular fluid of the oocyte).

RESULTS Pf o f CBCEPCs To monitor cell volume, we determined light-scattering intensity, as previously reported. 9 One of the experiments is depicted in detail (Fig. 1). At time zero, cells were superfused with a 10% hypotonic solution; the cells swelled rapidly, reaching a new steady state volume in approximately 10 seconds. The experimental data points were fitted to a curve, and from this curve the kinetic constant k, the apparent unstirred layer 8, and Pj were determined. Unstirred layer values were 80 ixm to 120 ju.m (not shown). Figure 1 shows examples of individual k and /^values, how 1 mM/XlMBS (preincubation: 20 minutes) slowed the osmotic response, and how, in the cells thus treated. 5 mM dithiothreitol (incubation: 5 minutes) subsequently restored the response almost completely. Average Pf values are shown in the inset to Figure 1. For control experiments, the characteristic osmotic kinetic constant k at 37°C was 0.52 ± 0.04 seconds" 1 (ji = 16), which translates into a Pfof 71.7 ± 6.2 jam/sec. In the same cells, after 20 minutes of preincubation with 1 mM pCMBS, the /^decreased to 26.2 ± 2.2 p.m/sec (;/ = 7). Finally, /JCMBS was removed by washing with isotonic solution containing 5 mM dithiothreitol; in the subsequent challenge, /^-increased to 62.7 ± 1.8 /xm/sec (•« = 5), for a recovety of 80%.

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20100 H2O CBCEPC mRNA oocytes injected with: FIGURE 3. Expression of water channels in batch 2. Oocyics were injected with water or cultured bovine cornea! epithelial cell (CBCEPC) poly(A + ) RNA, and the effects of nquaporin (AQP)I or AQP5 sense or antisense oliyonucleotides on the expressed /^ were determined. Bars, mean ± SE; numbers within bars are the number of oocytes in each group.

Measurement of Oocyte Volume and Osmotic Water Permeability Results of experiments in which oocytes were injected with water, CBCEPC poly(A l ) RNA, or AQPI cRNA are shown in Figure 2. The Pf of control oocytes injected with water was 14.3 — 1.8 ftm/sec (ri = 4). In oocytes injected with poly(A') RNA, /^increased to 76.7 ± 6.1 /xm/sec (n = 6). Previous studies have shown that HgCl2 inhibits the water permeability expressed in oocytes injected with cRNA encoding the water channel CH1P28 (or AQP1). I() " We incubated poly(A"h") RNA-injcctcd oocytes (« = 6) for I minute in 0.3 mM HgCl2. and found that their expressed water permeability was inhibited by 72% (decreased to 21.5 ± 0.7 jLtni/sec) compared with the control value. This inhibition was reversed when similarly treated oocytes were additionally incubated for 30 minutes with 5 mM j3-mercaptoethanol (/',- = 61.4 ± 7.6 jum/scc; n = 6).

Identification of an AQP5 Water Channel in CBCEPC mRNA To characterize the AQP message in CBCECPs, we coinjected oocytes with CBCEPC poly(A') RNA together with sense or antisense oligonucleotides for AQPI and AQP5. Coinjection of AQP5 antisense oligonucleotide inhibited this expressed Pf by 58% (Fig. 2), whereas no inhibitor^' effect occurred when coinjecting AQP5 sense oligonucleotide. As a control to verify the specificity of the AQP5 antisense probe used, we injected oocytes with AQPI cRJNA. As expected, the Pf rose drastically (Fig. 2). .Such expressed Pf was unaffected by AQP5 and AQPI sense oligonucleotides and by the AQP5 antisense probe under scrutiny but was inhibited by

90% by AQPI antisense oligonucleotide. confirming that there was no cross-reactivity between the AQP5 antisense probe and the AQPI cRNA. A similar control was performed injecting oocytes with CBCEPC mRNA. Because this batch of oocytes exhibited a slightly lower level of expression, these results are shown separately (Fig. 3). In this batch, AQP5 antisense once more inhibited the expressed /', (by 56%), whereas AQPI antisense resulted in a /Rvalue of 50.6 ± 0.9 (control value: 57.7 ± 4.7). The difference was not statistically significant (P = 0.08; /-test).

DISCUSSION The experiments in which light scattering was used to determine the CIJCEPCs Pf showed a comparatively high Pf value typical of cells that express water channels. The reversible block of such high osmotic permeability by a mercurial agent is also characteristic of the presence of functional water channels. The injection of cellular RNA in oocytes confirms that these cells express typical mercurial-sensitive functional water channels. The experiments with antisense oligonucleotides confirm that AQP5 is present in conical epithelium and strongly suggest that the predominant water channel message in CHCEPC poly(A + ) RNA is AQP5. In this regard, it is interesting to note that the AQP5 antisense did not inhibit the expressed Pf more than approximately 60%. which is consistent with the presence of additional water channel isoforms in these cells. The absence of effect of AQPI antisense oligonucleotide seems to exclude that isoform. In fact, after this manuscript was submitted, an article appeared showing immu-

10VS, January 1999, Vol. 40, No. 1 nocytochemical evidence for the presence of prominently expressed AQI'5 and weakly expressed AQP3 in corncal epithelium.12 In summan'. the presence of functional water channels in large numbers underlines that the corneal epithelium may contribute to maintain corneal hydration in a way more important than has been generally recognized so far.

References 1. Fischbarg J. The biology of ihc cornea. In: Bittar liF. ed. Fundamentals of Medical Cell Biology. Neurobiology, Thennobio/og)', and Cytobiology. Vol. 6. Greenwich, CT: JAI Press; 1992:151 -165. 2. Green K. Relation of epithelial ion transport to corneal thickness and hyclration. Nature. 1968:217:1074-1075. 3. Klyce SD. Transport of Na. Cl. and water by the rabbit corneal epithelium at resting potential. Am J I'bysiol. I975;228:14461452. 4. Candia OA. Fluid and Cl transport by the epithelium of the isolated frog cornea |Abstract|. Fed Proc. 1976:35:703. 5. Candia OA, Zamudio AC. Chloride-activated water permeability in the frog conical epithelium. J Membr liiol. 1995:143:259-266.

Lenticular Fluorescence as a Poisson Process Robert Alexander Weale determine whether published data on lenticular fluorescence can be described in terms of the Poisson statistic. PIJRIO.SK. TO

The intensity of the Poisson function is expressed in terms of estimates of the fraction of the incident light absorbed by the human lens. Functions calculated for ages 0 to 80 years for probabilities of different numbers of quanta absorbed are compared with experimental results. METHODS.

account for the observed data, a minimum of three exciting quanta (490 nm) absorbed has to be postulated for young lenses, but only two for older ones. At shorter wavelengths (380-450 nm), the tentative corresponding values are five and three, respectively.

RESULTS. TO

The model based on the age-related and wavelength-dependent changes in the minimum number of quanta absorbed from five to two is consistent with an age-related increase in the number of fluorophorcs and in their conformational changes. (Invest Ophtbalmol Vis Sci. 1999:40:257-260) CONCLUSIONS.

he fluorescence of the human eye lens has received a great deal of attention not only because of its intrinsic interest but also because it can play a disturbing role in human vi-

T

From the Age Concern Institute of Gerontology. King's College, London, United Kingdom. Submitted for publication January 26, 1998; revised May 5 and July 17, 1998: accepted August 21, 1998. Proprietary interest category: N. Reprint requests: Robert Alexander Weale, Age Concern Institute of Gerontology, Kings College London, Cornwall House, Waterloo Road, London SI: I 8WA, UK.

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6. Norwich T, Ibarra C, Ford P, Zamudio A, Parisi M. Candia OA. mRNA from frog corneal epithelium increases water permeability in Xenopus oocytes. Invest Ophtbalmol Vis Sci. 1995:36:27722774. 7. Raina S. Preston GM. Guggino \VP. Agre P. Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal and respirator)1 tissues../ liiol Chem. 1995;270:1908 -1912. 8. Cubbit I.C, Tang Q, Monteiro AC, Lausch IIR, Oakes EJ. Gene expression in cultures of human corncal epithelial cells and keratocytes. Invest Ophtbalmol Vis Sci. 1993:34:3199-3206. 9. FischbargJ, Li J, Kuang K, Echcvarria M, Iscrovich P. Determination of volume and water permeability of plated cells from measurements of light scattering. AmJ Pbysiol. I993;265:C14I2-C1423. 10. lichevarrfa M, Kuang K, Iscrovich P, et al. Cultured bovine corneal endothelial cells express CHIP28 water channels. Am J I'bysiol. 1993;265:CI349-CI355. 11. Preston GM, Carroll \VB, Guggino WH, Agre P. Appearance of water channels in Xenopus oocytes expressing red cell CIIIP28 protein. Science. 1992;256:385-387. 12. Hamann S, Zeuthen T, I.a Cour M, et al. Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye. AmJ I'bysiol. 1998;274:C1332-CI345.

sion.1'2 It is elicited in general by radiations in the shortwavelength part of the visible spectrum and, in accordance with Stokes' Law, manifests with emissions in its central part.2 The number of individual fluorophorcs is not yet known, due in part to incomplete stimulus control in past studies/ Several studies show that lenticular fluorescence increases approximately in proportion to age/~ s with the difference between different sets of data resting essentially on a scaling factor. There is some argument as to whether the relevant regressions* pass through zero. Departures from zero such as have been reported are based on the regressions themselves, and a negative intercept on the y axis must clearly result from fitting a linear regression to a set of data, convex toward the abscissa and passing through the origin. The above-mentioned rise in fluorescence and the welldocumented age-related increase in lenticular absorbance6 suggest that more fluorescent quanta are emitted with age because more exciting quanta are absorbed. However, a quantitative analysis fails to support this qualitative prediction7 and suggests, in accordance with an earlier suggestion,8 that some additional mechanism is likely to be involved: It is hypothesized that the conversion efficiency of the exciting quanta increases with age. The attempt is made here to see whether the Poisson statistic may help to elucidate an underlying process.

METHODS

Framework The approach is based on the following consideration: A't. = kf[[j{a) X A',]

(/)

where NK, the number of fluorescent quanta emitted, is a function of the number of quanta incident on a molecule Nt and p(a), the probability of their being absorbed, and k is a scaling constant.