JOURNAL OF CELLULAR PHYSIOLOGY 143:455-459 (1990)

Volume Regulation in Lens Epithelial Cells and Differentiating Lens Fiber Cells DAVID C. BEEBE,* JUDITH T. PARMELEE, AND KARLA S. BELCHER Department of Anatomy, Uniformed Services University of the Health Sciences Bethesda, Maryland 208 74-4799 (D.C.B., K.S.B.); Department of Clinical Neurosciences, Brown UniversitylRhode Island Hospital Providence, Rhode Island 02903 (1.J.P.) Previous studies from our laboratory have led us to conclude that lens cell elongation is caused by an increase in cell volume. This volume increase results from an increase in the potassium content of the cells due to decreased potassium efflux. In contrast, an increase in the volume of most cells triggers a regulatory volume decrease (RVD) that i s usually mediated by increased potassium efflux. For this reason, chicken embryo lens epithelial cells were tested to see whether they were capable of typical cell volume regulation. Changes in cell volume during lens fiber differentiation were first estimated by 3H,0 water uptake. Cell water increased in proportion to cell length in elongating lens cells. Treatment of epithelial cells cultured in basal medium with dilute or concentrated medium, or with medium containing 50 m M sucrose, resulted in typical volume regulatory responses. Cells lost or gained volume in response to osmotic stress, then returned to their previous volume. In addition, the elongation and increase in cell volume that accompanies lens fiber cell differentiation occurred normally in either hypoor hypertonic media. This observation showed that the activation of mechanisms to compensate for osmotic stress did not interfere with the increase in volume that accompanies elongation. The ability of elongating cells to volume regulate was also tested. Lens epithelial cells were stimulated to elongate by exposure to embryonic vitreous humor, then challenged with hypotonic medium. These elongating cells regulated their volume as effectively as unstimulated cells. Therefore, cells that were increasing their volume due to reduced potassium efflux could adjust their volume in response to osmotic stress, presumably by increasing potassium efflux. This suggests that the changes in potassium efflux that occur during differentiation and RVD are regulated by distinct mechanisms.

The eye lens consists of two cell types. A monolayer of cuboidal epithelial cells covers the anterior lens surface, and highly specialized fiber cells make up the body of the lens. Lens fiber cells differentiate from the epithelial cells at the equator of the lens, a process that continues throughout life. Although the epithelial cells in the center of the lens do not normally form fibers, central epithelial cells from chicken embryos can be induced to form fiber-like cells in vitro (Philpott and Coulombre, 1965).We have previously identified a protein in the vitreous humor of the eye, called lentropin, which promotes lens fiber cell differentiation (Beebe et al., 1980). Lentropin is functionally and immunologically related to the family of insulin-like growth factors (IGF). When explanted sheets of embryonic chicken lens epithelial cells are exposed to vitreous humor or IGF-1, one can study lens fiber cell formation under controlled conditions (Beebe et al., 1987). The transformation of lens epithelial cells into fibers involves major changes in morphology and metabolism. The cells stop dividing, elongate to more than 50 times their original length, and lose their intracellular membrane-bound organelles. Fiber cells synthesize several specialized membrane proteins and accumulate large quantities of lens-specific proteins, the crystal0 1990 WILEY-LISS, INC.

lins. Studies on the mechanisms of lens fiber cell elongation indicate that this process is driven by an increase in cell volume. A model has been developed to show how an increase in volume is translated into cell elongation, both in culture and in the intact lens (Beebe et al., 1982). The cell volume increase seen during lens fiber cell differentiation contrasts with the tendency of most cells to maintain their volume within narrow limits. We became interested in the volume regulatory ability of elongating lens epithelial when we found that the increase in cell volume that drives lens fiber cell elongation was due t o a reduction in the rate of potassium efflux (Parmelee and Beebe, 1988). It seemed, therefore, that both volume-regulatory mechanisms and the volume increases associated with fiber formation might be dependent on changes in ion transport across the cell membrane. The studies described in this paper were designed to examine the volume-regulatory ability of cultured lens epithelial cells, both before and during lens fiber differentiation. Received December 5, 1989; accepted February 2, 1990.

*To whom reprint requestslcorrespondenceshould be addressed.

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MATERIALS AND METHODS Water uptake experiments Fertile chicken eggs were purchased from Truslow Farms (Chestertown, MD) and incubated a t 38°C in a Humidaire model 55 forced draft incubator. Central lens epithelia from 6-day-old chicken embryos were explanted according to previously published methods (Philpott and Coulombre, 1965; Piatigorsky, 1975; Beebe and Feagans, 1981) and incubated in either vitreous humor or Ham’s F-10 medium. Cytochalasin B was prepared as a concentrated stock in dimethylsulfoxide (DMSO), then further diluted with medium to the appropriate concentration. DMSO was added to the relevant controls in like concentration, with no measurable effect on water content in either stimulated or unstimulated explants. The explanted epithelia were maintained in a Forma Scientific tissue culture incubator a t 37°C in a 95% air/5% CO, atmosphere for the times indicated in Results. Water content was measured by transferring a single freshly dissected or previously incubated lens epithelium to a conical microcentrifuge tube, removing any excess medium and incubatin in 10 p1 of Hams F-10 medium containing 1.0 pCi of 5H,O and 1.0 pCi of 14Cinulin for 5 min. Preliminary experiments showed that ’H,O labeling plateaued by 2 min of incubation; thus 5 min incubations ensured maximum specific activity of accessible intracellular water. After isotopic incubation, 0.25 ml of a silicone oil/hexane mixture having a calculated density of 1.01, slightly greater than that of the medium, was layered over the explant and labeled medium in the bottom of the tube (Fig. 1).Immediately after adding the oil mixture, the tube was centrifuged in an Eppendorf microcentrifuge for 30 sec. This resulted in the rapid separation of the explant from the medium (Fig. 1).Three microliters of the incubation solution was removed from the drop near the top of the oil and diluted in 0.5 ml of 1%sodium dodecyl sulfate (SDS).The silicone oil and the remaining medium were removed and discarded and the explant, which was flattened on the bottom of the tube, was dissolved in 0.5 ml 1%SDS. Medium and tissue radioactivity were measured in a dual-channel Beckman LS-9000 liquid scintillation counter. Using the 14Ci3Hratio of the medium and the 14C counts per minute (cpm) associated with the explant, the cpm of water in the extracellular

TABLE 1. Water content of untreated, vitreous humor-treated and cytochalasin-treated6 day lens epithelial Incubation medium Ham’s F-10 Vitreous Vitreous + cytochalasin B (5 pg/ml)

n 12 14 11

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‘Incubationtime prior to volume determination was one hour. Steady-state water content was measured after incubation with ’H20 for 5 min. Values are means 5 SE. ‘Significantly different (P< 0.05) from explants cultured in Ham’s F-10 medium or in vitreous humor + cytochalasin B. 3Not significantly different from explants incubated in Ham’s F-10 medium.

space of the explant was calculated. This was subtracted from the total 3H counts in the explant. The remaining counts were converted to nanoliters of water by calculation from the 3H-specific activity of the medium (cpm/pl).Quench correction was unnecessary, because the addition of an equal volume of SDS to the small samples provided constant quench. Elongation experiments Dissection and incubation were carried out as described above. In some experiments the osmolarity of the culture medium was increased by the addition of 50 mM sucrose or an amount of 10 x -concentrated Ham’s F-10 to increase osmolarity 50% above normal. In experiments testing the effect of hypotonic medium, distilled water was added to the normal-strength Ham’s F-10 medium to decrease osmolarity by 25%. Cell lengths were determined on a Zeiss inverted microscope by the optical method of Beebe and Feagans (1981). Each explant was measured at its center and at four equally spaced locations 125 pm from the center.

Solutions and drugs Tissue culture medium was obtained from GIBCO (Grand Island, NY). 3H-waterwas purchased from New England Nuclear (Boston, MA) and 14C-inulin from ICN Radiochemicals (Irvine, CAI. Silicone oil (high temperature) was from Aldrich Chemical Co. (Milwaukee, WI). All other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Vitreous humor extract was prepared from 15-day-oldchicken embryos as previously described (Beebe et al., 1980; Beebe and Feagans, 1981) and buffered by the addition of sodium bicarbonate (1.2 mg/ml). Statistics Determinations of significant difference were based on Student’s t test.

RESULTS Water accumulation during elongation Lens epithelia explanted in Ham’s F-10 with no differentiation stimulus had an average intracellular aqueous volume of 18 nl/explant as measured by the tritiated water technique (Table 1;Fig. 2, time 0). One hour after stimulation with vitreous humor this volume increased to 25 nl. The values for 5 and 24 hr of incubation in vitreous-supplemented medium were 30 and 80 nl, respectively (Fig. 2). Cytochalasin B and D were previously shown to prevent elongation in this

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TIME (hr) Fig. 2. Water accumulation during elongation of lens epithelial cells from 6 day chicken embryos. Explants were stimulated with vitreous humor for the times shown. Aqueous volume was determined by incubating with 3H,0 for 5 min. Error bars fell within the symbols. Number of determinations ranged between 17 and 35 per data point.

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system (Piatigorsky et al., 1972; Beebe and Cerrelli, 1989). Lens epithelia cultured for 1 hr in vitreous humor containing 5 pg/ml of cytochalasin B had an average water content of 20 nl (Table 1). This was not significantly different from the aqueous volume of unstimulated cells but was significantly less than the volume of cells cultured in vireous humor without cytochalasin (P < 0.05).

Volume regulation Lens epithelia incubated for 1 hr in hyper- or hypotonic culture medium appeared to maintain normal length (results not shown). This was studied more closely by exposing freshly explanted lens epithelia to dilute or concentrated medium and recording changes in cell length a t 3 min intervals. Cells exposed to the diluted medium elongated from an original length of 10.5 pm to 12.3 km in 6 min. They returned to their original length by 15 min (Fig. 3A). Cells exposed to 1.5 x -concentrated medium decreased in length from 11.7 pm to 8.1 pm within 3 min and nearly regained their original length by 15 min (Fig. 3B). Explants exposed to medium made hypertonic with added 50 mM sucrose also decreased and subsequently regained length, but over a much longer time; cells originally 10.4 pm decreased to 8.8 pm in 15 min and returned to 10.2 pm after 1 hr (Fig. 3C). The differences between beginning cell lengths and the maximum or minimum length induced by these treatments was significant at P < 0.01 in each case. Thus, the apparent lack of change in cell length in unstimulated epithelia 1 hr after exposure to hypo- or hypertonic medium was due to the completion of volume regulation during this period. Epithelial cells that were stimulated to differentiate with vitreous humor for 10 min, then exposed to hypoor hypertonic vitreous, elongated like those continuously exposed to unmodified vitreous (Fig. 4). Cells cultured in normal-strength vitreous humor were between 18 and 20 pm long after 6 hr of culture. Epithelia cul-

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TIME [ m i d Fig. 3. Cell volume regulation in 6-day-old embryonic chicken lens epithelia. Explants were exposed to A: hypotonic solution (osmolarity decreased 25%with distilled water), B hypertonic solution (osmolarity increased 50% with concentrated Ham’s F-10 medium), or C: hypertonic solution (50 mM sucrose added). Points are mean SE. At points where error bars are not displayed, they fell within the symbol. Number of determinations per data point ranged from 15 to 36. Stars indicate lengths that differed significantly (P < 0.01) from the initial values.

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tured in dilute vitreous elongated a t virtually the same rate as controls (Fig. 4A). Those exposed to 1.5 x -concentrated medium always averaged about 1 pm shorter than controls (Fig. 4B), although these differences were not statistically significant. Normal elongation was also seen in cells incubated in medium made hypertonic with 50 mM sucrose (Fig. 4C). In a separate experiment, cells were cultured in vitreous humor for 1.5 hr, then sufficient distilled water was added to dilute the vitreous to 75% strength. This treatment tested whether elongating cells were able to volume regulate in a manner similar to that seen in unstimulated cells (Fig. 3). At the time of exposure to hypotonic medium, cells averaged 15.0 pm in length. Within 3 min of diluting the medium, cell length increased to 19.0 pm. By 12 min, cells returned to their previous length (Fig. 5).

DISCUSSION Cell volume regulation has been extensively studied in several cell types (recent reviews; Eveloff and Warnock, 1987; Hoffmann and Simonsen, 1989). In general, if cells are exposed to hypertonic medium, they initially lose water as a consequence of the difference in osmotic pressure across the cell membrane. This shrinkage stimulates NaCl uptake, which is followed by osmotic water influx. This restores normal cell volume. This mechanism has been called regulatory volume increase

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Fig. 5. Volume regulation during lens fiber cell elongation. Lens epithelia were stimulated with vitreous humor for 1.5 hr, a t which time the medium was diluted with distilled water to 75% of normal osmolarity. Cell length was determined every 3 min for 15 min. Points are means -C SE. Stars indicate cell lengths that were significantly different (P< 0.01) from the length of the cells just before dilution of the medium.

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INCUBATION TIME (hrl Fig. 4. Cell volume regulation in differentiating 6-day-old embryonic chicken lens epithelia. Explants were stimulated to elongate with vitreous humor. Controls are represented by triangles. Circles represent cell lengths in solutions made hypotonic (A) or hypertonic (B,C) as in Figure 3. Error bars indicate SEM. Number of determnations per data point ranged from eight to 40. There were no significant differences between any of the treated explants when compared to controls.

(RVI).In the opposite case, exposure of cells to hypotonic medium initially results in water uptake. This is followed by regulatory volume decrease (RVD),which usually involves loss of intracellular KC1 with consequent water efflux, which restores cell volume. Earlier studies on whole lenses have shown that they volume regulate by mechanisms that resemble those used by cultured cells. Rat lenses cultured in hypotonic medium increased in volume, then lost potassium to return to their initial volume (Patterson and Fournier, 1976). Similarly, sodium uptake was the primary mediator of RVI when rat lenses were cultured in hypertonic medium (Patterson, 1981a). Neither RVI nor RVD was prevented by ouabain in these studies (Patterson, 1981b). The aqueous volume determinations shown in Table 1 and Figure 2 verified volume measurements previously assessed by microscopic methods (Beebe et al., 1979). These data also showed that volume increase will not occur if elongation is blocked with cytochalasin (Table 1). We have shown that volume increase and cell elongation are driven by potassium accumulation (Parmelee and Beebe, 1988). Cytochalasin prevents potassium accumulation by increasing potassium efflux (Beebe and Cerrelli, 1989). Comparison of initial changes in cell volume and length showed that these values were directly propor-

tional. The length of unstimulated cells was approximately 11pm (Fig. 3) and vitreous-stimulated explants after 1 hr were about 15 bm long (Fig. 5). The cell volumes at these times were 18 and 25 nl, respectively (Table 1).Thus, cell length and volume increased 3540% in the initial stage of elongation. We have previously argued that the physical constraints of lens epithelial organization require that if volume increases, the cells must elongate to a similar extent (Beebe et al., 1982). The cell volumes determined here also confirm the validity of cell length measurements as a measure of volume change in this system. Experiments with lens epithelial explants showed that application of hypertonic or hypotonic medium resulted in an initial osmotic volume change, with subsequent return to normal (Fig. 3). That is, embryonic lens cells appeared to react with a classic volume regulatory response to osmotic perturbation. Differentiating epithelial cells were able to elongate in spite of continued osmotic stress (Fig. 4). This demonstrates that elongation depends on active volume expansion, because cell volume increased in spite of an increase in extracellular solute concentration. In addition, dilution of the external medium did not cause more rapid elongation. This suggests that volume regulation occurred in elongating cells. The data presented in Figure 5 showed directly that differentiating lens cells can volume regulate, even while increasing in volume. By 1.5 hr after exposure to vitreous humor, the cells had already elongated from about 11 pm to 15 pm. At this time the cells responded to dilution of the medium with a brief increase in cell length, followed by a return to their length before the osmotic shock. When these data are combined with the results shown in Figure 4A, it is evident that, when differentiating cells were stimulated to elongate in hypotonic

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medium, they swelled, then immediately decreased their volume in the same way as unstimulated cells. They then went on to increase their volume as a consequence of differentiation. Thus, while potassium efflux is suppressed to increase cell volume (Parmelee and Beebe, 19881, potassium efflux may be transiently increased for RVD. These responses involve opposing changes in potassium flux and cell volume. Apparently, lens fiber elongation and volume regulation are sensitive to different signals and operate through different transduction pathways. The biochemical events that mediate the effect of lentropin on lens epithelial cells are largely unknown. Zelenka and coworkers (1982) have shown that a transient increase in the methylation of phosphatidylethanolamine (PE) is an early event in fiber cell differentiation. In addition, inhibition of phospholipid methylation prevented cell elongation. Thus increased methylation of PE may be part of the pathway that decreases potassium efflux during lens fiber differentiation. The mechanisms that activate the ion transporters in volume regulation are also not clearly defined. However, there is evidence that the phosphoinositide pathway (Grinstein et al., 1985, 1986) and/or changes in intracellular calcium activity (Davis and Finn, 1987; Foskett and Spring, 1985; Hoffmann, 1986; Wong and Chase, 1986)may have roles in these responses in some cell types. In summary, our results demonstrate that efficient volume-regulating mechanisms exist in lens epithelial cells, both before and during their differentiation into fibers. The elongating cells can increase their volume in response to factors that trigger differentiation and, at the same time, regulate their volume in response to osmotic challenge. Further experiments will be needed to identify the pathways that control both the volume increase and the volume regulation of developing lens fibers.

ACKNOWLEDGMENTS Support was provided by NIH grant EY04853. The opinions or assertions in this paper are the private ones of the authors and are not to be construed as official or reflecting the views of the DoD or the USUHS. The experiments reported were conducted according to the principles set forth in the “Guide for Care and Use of Laboratory Animals,’’ Institute of Laboratory Animal Resources, National Research Council (DHEW Pub. NO.NIH 78-23).

LITERATURE CITED Beebe, D.C., and Cerrelli, S. (1989) Cytochalasin prevents cell elongation and increases potassium efflux from embryonic lens epithelial cells: Implications for the mechanism of lens fiber cell elongation. Lens Eye Tox. Res., in press. Beebe, D.C., Compart, P.J., Johnson, M.C., Feagans, D.E., and Feinberg, R.N. (1982) The mechanism of cell elongation during lens fiber cell differentiation. Dev. Biol., 92:54-59. Beebe, D.C., and Feagans, D.E. (1981) A tissue culture system for studying lens cell differentiation. Vision Res., 21 :113-118. Beebe, D.C., Feagans, D.E., Blanchette-Mackie, E.J. and Nau, M.E. (1979) Lens epithelial cell elongation in the absence of microtubules: evidence for a new effect of colchicine. Science, 206:836-838. Beebe, D.C., Feagans, D.E., and Jebens, H.A.H. (1980) Lentropin: A factor in .vitreous humor which oromotes lens fiber cell differenti. ~ ~ ~~.~~ ~~~.~ ation. Proc. Natl. Acad. Sci. USA, 77:490-493. Beebe, D.C., Silver, M.H., Belcher, K.S., Van Wyk, J.J., Svoboda, M.E..and Zelenka, P.S. (1987) Lentropin, a protein that controls lens fiber formation, is related functionally arid immunologically to the insulin-like growth factors. Proc. Natl. Acad. Sci. USA, 84: 2327-2330. Davis, C.W., and Finn, A.L. (1987) Interactions of sodium transport, cell volume and calcium in frog urinary bladder. J . Gen. Physiol., 89:687-702. Eveloff, J.L., and Warnock, D.G. (1987) Activation of ion transport systems duringcell volume regulation. Am. J . Physio1.,252:Fl-F10. Foskett, J.K., and Spring, K.R.(1985) Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation. Am. J. Physiol., 248:C27-C36. Grinstein, S.,Cohen, S., Goetz, J.D., and Rothstein, A. (1985) Osmotic and phorbol ester-induced activation of Na + / H +exchange: Possible role of protein phosphorylation in lymphocyte volume regulation. J. Cell Biol., IOIt269-276. Grinstein, S., Goetz-Smith, J.D., Stewart, D., Beresford, B.J., and Mellors, A. (1986) Protein phosphorylation during activation of Na+/H+ exchange by phorbol esters and by osmotic shrinking. J. Biol. Chem., 261r8009-8016. Hoffmann, E.K. (1986) Anion transport systems in the plasma membrane of vertebrate cells. Biochim. Biophys. Acta., 864:l-31. Hoffmann, E., and Simonsen, L. (1989) Membrane mechanisms in volume and pH regulation in vertebrate cells. Physiol. Rev., 69: 315-382. Parmelee, J.T., and Beebe, D.C. (1988) Decreased membrane permeability to potassium is responsible for the cell volume increase that drives lens fiber cell elongation. J . Cell. Physiol., 134:491-496. Patterson, J.W. (1981a) Lens volume regulation in hypertonic medium. Exp. Eye Res., 32:151-162. Patterson, J.W. (1981b) The effect ofouabain on volume regulation in the rat lens. Invest. Ophthalmol. Vis. Sci., 20:40-46. Patterson, J.W., and Fournier, D.J. (1976) The effect of tonicity on lens volume. Invest. Ophthalmol. Vis. Sci., 15:866-869. Philpott, G.W., and Coulombre, A.J. (1965) Lens development 11. The differentiation of embryonic chick lens epithelial cells in vitro and in vivo. Exp. Cell Res., 383335444, Piatigorsky, J . (1975) Lens cell elongation in vitro and microtubules. Ann, N.Y. Acad. Sci., 253:333-347. Piatigorsky, J.,Webster, H. de F., and Wollberg, M. (1972) Cell elongation in the cultured chick lens epithelium with and without protein synthesis. J . Cell Biol., 55r82-92. Wong, S.M.E., and Chase, H.S. Jr. (1986) Role of intracellular calcium in cellular volume regulation. Am. J . Physiol., 250tC841-C852. Zelenka, P.S., Beebe, D.C., and Feagans, D.E. (1982) Transmethylation of phosphatidylethanolamine: An initial event in embryonic chicken lens fiber cell differentiation. Science, 21 7:1265-1267. ~~

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