Exp. Eye Res. (1991)

53, 681-684



Influence of Bicarbonate and Insulin on Pigment Synthesis Cultured Adult Human Retinal Pigment Epithelial Cells It is usually assumed that the adult retinal pigment epithelium. (RPE) of humans and other mammals has negligible capacity to synthesize melanin granules. Consistent with this idea is the observation that human RPE cells that have divided extensively in culture lose endogenous pigment by dilution and do not synthesize new pigment in vitro (Flood, Gouras and Kjeldbye, 1980). However, adult lapine (Laties and Lerner, 1975) and bovine (Dryja et al., 1978) RPE contains tyrosinase activity, and RPE cultured from 4-6-month-old pigs synthesizes pigment granules in vitro (Dorey, Torres and Swart, 1990). Recently, during studies in this laboratory unrelated to pigment synthesis, cultures of aged adult human RPE maintained with Dulbecco’s Modified Eagle’s (DME) medium supplemented with insulin were observed to contain foci of newly divided cells that became intensely pigmented. Pigment synthesis by adult human RPE cultured in a DME-based medium has also been observed by others (C. K. Dorey, pers. commun.). Since de novo pigment synthesis by RPE cultured from aged human donors had not previously been observed with a number of other media [minimum essential medium (MEM), medium 199, Ham’s F- 10 nutrient solution, or PM medium ; Flood et al., 1980 ; Edwards and Kurtz, unpubl. ohs.], the result with DME supplemented with insulin prompted experiments to define more precisely the medium requirements for pigment synthesis. A possibly related observation is the report that increased bicarbonate levels led to earlier pigment synthesis by B16 melanoma cells (Laskin et al., 1980). In this report it is, shown that cultured human RPE from aged donors synthesize pigment when maintained in DME but not in other media, that an elevated bicarbonate level is a required feature of DME for this synthesis under the conditions of these experiments, and that insulin added to DME significantly shortens the in vitro time required for this synthesis. Human autopsy eye of donors 64 or more years of age were obtained through the National Disease Research Interchange, Philadelphia. RPE cells were isolated as previously described (Edwards, 1982) suspended in PM medium (Pfeffer et al., 1986 ; Edwards, Adler and Claycomb, 1991) that included 10 pug ml-’ bovine insulin (Collaborative Research, Bedford, MA) and 20 pg ml-’ gentamicin, and seeded at 5 or 10 x lo3 cells cm+ in 4-cm2 12-cluster wells (Costar, Cambridge, MA) or in 25-cm2 flasks (Falcon, Oxnard, CA). Additional cells were seeded at the same densities in 96-cluster wells for immunocytochemical 00144835/91/l




staining for cytokeratins (Leschey et al., 1990). Medium was changed every 34 days ; volumes of medium were 0.5 ml for the 12-cluster wells and 3 ml for the flasks. Cultures were maintained at 37°C in a water-saturated atmosphere of 5 y0 CO,/9 5 y0 air. Cells were allowed to proliferate until the cultures were confluent (usually l-2 weeks), at which time PM medium was replaced with one of the following experimental media, all of which were supplemented with 10% (by volume) fetal bovine serum and 20 pg ml-’ gentamicin: (1) DME (which contains 44 mM bicarbonate and 1 g 1-l); (2) DME with bicarbonate reduced to levels of the other media (26.2 mM) and the omitted bicarbonate replaced with 17.8 mM NaCl : (3) MEM ; (4) MEM supplemented with extra tyrosine to equal the tyrosine concentration of DME (0.40 mM compared to 0.22 mM of MEM) ; (5) Medium 199 : or (6) DME supplemented with 10 pg ml-’ insulin. Media were obtained as bicarbonate-free powder (Grand Island Biological Company, Grand Island, NY). A total of six 12-cluster cultures were tested with each medium for each of two donors, three each at both cell densities (5 or 10 x lo3 cells cmm2). At least one 25-cm3 flask was also tested for each medium for the two donors. In addition, confluent cultures from a 72-year-old female donor were subcultured with trypsin (10: 1 split), grown to confluency, and tested with the six experimental media. In vitro incubation times refer to the time in culture after the switch to one of the experimental media. Unstained cultures were photographed with a phase-contrast microscope, or with bright-field optics when it was desired to show only opaque material such as newly synthesized pigment. Prior to switching cultures to one of the six experimental media, confluent cultures of adult human RPE cells were largely devoid of pigment as a result of extensive cell division. Cells maintained for 4 weeks or longer in DME supplemented with 10 % fetal bovine serum contained foci of newly pigmented cells. Figure 1 shows cells maintained in DME for 6 weeks. When viewed with bright-field optics ([Fig. l(B)], cells that had not yet synthesized pigment were nearly invisible except for the cell borders [Fig. l(B) right side], while other cells contained varying amounts of opaque pigment [Fig. l(B) left side]. Cultures maintained for as long as 4 months in MEM or medium 19 9 contained cells that were of similar morphology to the cells of Fig. 1 but never contained areas with newly 0 1991 Academic Press Limited


FIG. 1. Repigmentation in RPE cells from a 72-year-old female maintained for 6 weeks in DME plus 10% serum. A, Cells viewed with phase-contrast optics. B, Same field viewed with bright-field optics, showing only opaque pigment. The arrow in each part of the figure indicates three adjacent cells that appear not to have divided in culture and to contain pigment that was present before isolation of the RPE. x 16 5.

FIG. 2. Cultures of RPE from the same donor as in Fig. 1 maintained for I I weeks in one of the following media, all supplemented with 10% fetal bovine serum and 20 rug ml-’ gentamicin: (1) DME (with 44 mM bicarbonate): (2) DME with bicarbonate reduced to 26.2 mM and the omitted bicarbonate replaced with 17.8 mM NaCl: (3) MEM: (4) MBM with 0.40 mM tyrosine ; (5) Medium 199 ; and (6) DME with 10 pug ml-’ insulin. Repigmentation occurred only in the DME-based media with high bicarbonate (media 1 and 6), and was more advanced in DME with insulin (medium 6).

synthesized pigment. Newly synthesized pigment could be distinguished from endogenous pigment that was

present before the cells had divided: the newly




FIG. 3. RPE from a 76-year-old female maintained in DME plus 10 /Lg ml-’ insulin for 32 weeks, at which time the culture was fixed in 10% formalin and photographed with bright-field optics to show only the opaque pigment. Much of the pigment is localized in discrete granules. x 730.

synthesized pigment was initially very fine and was nearly always present in many contiguous cells [Fig. l(B) left side], while pigment present before cell proliferation was usually granular or clumped and present in one or a few cells separated by cells devoid of pigment (Fig. 1, arrow). The newly synthesized pigment was not fluorescent when the cells were viewed with a microscope equipped with epifluorescence optics, whereas undivided cells with endogenous pigment were intensely fluorescent (data not shown). This indicates that the newly synthesized pigment was not lipofuscin. which is fluorescent (Feeney, 1978: Dorey et al.. 1990). Cultures maintained in DME contained newly pigmented areas that were visible with the unaided eye (Fig. 2, medium 1) and increased in pigmentation with time. In cultures that had been synthesizing pigment for some time, pigment was often present in the form of discrete granules (Fig. 31. Elevated bicarbonate concentration compared to other media is a feature of DME that was required for repigmentation under the conditions of these experiments. When the bicarbonate concentration of DME was reduced from 44 mM to 26.2 mM, repigmentation did not occur during periods of up to 4 months (medium 2, Fig. 2). The pH of the DME-based media that contained 44 mM bicarbonate (media 1 and 6, Fig. 2) was 0.2-03 pH units higher than the other experimental media when measured after 3-4 days of incubation with cultured RPE.






Another difference between DME and the other experimental media is a tyrosine concentration of 0.40 mM compared to 0.22 mM or less. MEM with 040 mM tyrosine did not promote the synthesis of visible pigment during incubations of up to 4 months, based on observations with the naked eye (medium 4, Fig. 2) and with the light microscope. Insulin added to the medium significantly shortened the time in vitro required for the appearance of visible repigmentation. Foci of pigment first became visible at about 2 weeks after the cultures were switched to DME supplemented with 10 pugml-’ insulin (medium 6, Fig. 2), i.e. in about half the time compared to DME without added insulin. With additional time in culture, the RPE maintained in DME with added insulin always contained more pigment than cultures in DME without added insulin, based on non-quantitative assessment with the naked eye. These results were obtained with all replicate cultures from a 64-year-old male and a 72-year-old female donor. First-passage cells from the 72-year-old donor also gave similar results: pigmentation of subcultured cells maintained in DME was visible with the naked eye at 7 weeks, while pigmentation of cells maintained in DME with added insulin became visible at 3 weeks. Cells that had proliferated in culture from both donors contained filaments that stained positively for cytokeratins (data not shown), indicating that the cells were of epithelial origin (compare with Leschey et al., 1990). In a preliminary experiment in which cultures were maintained with DME plus added insulin for 32 weeks, individual foci of heavily pigmented cells occasionally became necrotic in appearance and detached from the culture surface. McQuillan, Hutchinson and Panasci (1989) also observed that melanoma cells which become pigmented in culture did not remain viable. while replicate cultures maintained in medium with low tyrosine (011 mM) did not become pigmented and remained viable. It has not yet been determined if pigment synthesis by cultured adult human RPE cells is consistently associated with accelerated cell death. These results provide the first indication that bicarbonate influences pigment formation by the RPE and insulin influences pigment formation in any cell type. Considering first the effects of bicarbonate, it is clear that a concentration of this substance higher than 26.7 mM is a necessary feature of DME for repigmentation under the conditions in which these experiments were done; when bicarbonate was reduced from 44 to 26.2 mM in DME, repigmentation did not occur. [Note that physiological levels of bicarbonate are in the range of 18-27 mM. e.g. Bito. 1982.1 In B 16 melonoma cells the time required for melanogenesis is shortened by increased levels of bicarbonate (Laskin et al.. 1980). The RPE is known to respond to changes in extracellular bicarbonate concentrations in a number


of ways. The increase in extracellular pH seen in these experiments (0.2 pH units) is presumably accompanied by an increase in intracellular pH (e.g. Keller et al., 1986). In intact RPE-choroid preparations. decreases in extracellular bicarbonate concentrations are associated with reductions of chloride ion flux in the retina-to-choroid direction (Miller and Steinberg, 19 77a). a decrease in the transepithelial potential, and a depolarization of the apical membrane (Miller and Steinberg, 1977b ; Steinberg. Miller and Stern, 19 78 ; Keller et al., 1986). It is not known if any of the foregoing phenomena are causally related to increases in pigment synthesis associated with increased bicarbonate levels. Elevations of extracellular bicarbonate above 30 mM are also associated with a marked increase in the disk shedding response of isolated Xenopus eyecups (Besharse and Dunis, 1983). Insulin added to DME reduced the time required for the appearance of newly synthesized pigment, but pigment synthesis still occurred in DME without added insulin. Since all media were supplemented with 10% serum, which is known to contain insulin and insulinlike activities, conclusions cannot be drawn about whether or not insulin is required for pigment synthesis. Further tests with serum-free media are required to clarify the requirement of insulin for pigment synthesis. It is also not clear if insulin only results in an earlier initiation of pigment synthesis or if insulin also acts to increase the rate of synthesis. Other effects of insulin on cultured RPE include stimulation of DNA synthesis (Leschey et al., 1990) and preservation of retinyl ester synthesis (Edwards et al., 1991). The lack of effect of 0.40 mM tyrosine when combined with MEM shows that the concentration of tyrosine in DME is not, of itself, sufficient to cause pigment synthesis by cultured human RPE. This is noteworthy, since tyrosine is the substrate for the ratelimiting step in melanin synthesis, and tyrosine stimulates tyrosinase activity and pigment synthesis in cultured hamster melanoma cells (Slominski et al.. 1988). It is not known if the de novo pigment synthesis reported here is accompanied by increases in tyrosinase activity or amounts of melanin. Whether or not 0.40 mM tyrosine is necessary for pigment synthesis in combination with other components of DME will require experiments in which the tyrosine concentration of DME is varied. The synthesis of pigment by cells that had proliferated extensively in primary culture and by firstpassage cells indicates that human RPE cells from aged donors retain this differentiated property in spite of extensive cell division in vitro. Subcultured porcine RPE also synthesizes pigment (Dorey et al., 1990). The effects of bicarbonate and insulin may serve as a basis for additional studies on the mechanisms involved in pigment formation. Such studies may provide useful insights into the control of expression of differentiated properties of RPE.



Acknowledgements Autopsy human eyes were provided by the National Disease Research Interchange, Philadelphia, PA. This work was supported by NIH grant EY02028 (R.B.E.), the Boston University Advisory Committee on Research (M. J. K.), and unrestricted grants to the Department of Ophthalmology from the Massachusetts Lions Eye Research Fund, Inc., and Research to Prevent Blindness, Inc.

MARY J. KURTZ” ROSS B. EDWARDS” College of Basic Studies, Boston University, and a Department of Ophthalmology, Boston University School of Medicine, Boston, MA, U.S.A. *For reprint requests at: College of Basic Studies, Boston University, 871 Commonwealth Avenue, Boston. MA 02215, U.S.A.

References Besharse, J. C. and Dunis, D. A. (1983). Rod photoreceptor disc shedding in eyecups: relationship to bicarbonate and amino acids. Exp. Eye Res. 36, 567-80. Bito. L. Z. (1982). Ionic composition of ocular fluids. In Handbook of Neurochemistry, 2nd edn, Vol. 8. (Ed. Lajtha, A.). Pp. 477-506. Plenum Press: New York. Dorey. C. K.. Torres, X. and Swart, T. (1990). Evidence of melanogenesis in porcine retinal pigment epithelial cells in vitro. Exp. Eye Res. 50, l-10. Dryja, T. P., O’Neil-Dryja, M.. Pawelek, J. M. and Albert, D. M. (1978). Demonstration of tyrosinase in the adult bovine uveal tract and retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 17. 51l-14. Edwards, R. B. (1982). Culture of mammalian retinal pigment epithelium and neural retina. Methods Enzymol.

81. 3943. Edwards, R. B., Adler, A. J. and Claycomb. R. C. (1991). Requirement of insulin or IGF-1 for the maintenance of retinyl ester synthetase activity by cultured retinal pigment epithelial cells. Exp. Eye Res. 52, 51-7. (Received

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and accepted



Feeney, L. (19 78). Lipofuscin and melanin of human retinal pigment epithelium ‘: fluorescence, enzyme cytochemical and ultrastructural studies. Invest. Ophthalmol. Vis. Sci. 17. 583-600. Flood, M. T.. Gouras, P. and Kjeldbye. H. (1980). Growth characteristics and ultrastructure of human retinal pigment epithelium in vitro. Invest. Ophthalmol. Vis. Sci. 19. 1309-20. Keller. S. K.. Jentsch, T. J.. Koch, M. and Wiederholt, M. (1986). Interactions of pH and K+ conductance in cultured bovine retinal pigment epithelial cells. Am 1. Physiol. 250, C124-37. Laskin. J. D.. Mufson, R. A., Weinstein. 1. B. and Engelhardt, D. L. ( 1980). Identification of a distinct phase during melanogenesis that is sensitive to extracellular pH and ionic strength. 1. Cell. BioZ. 103. 467-74. Laties, A. M. and Lerner. A. B. (1975). Iris colour and relationship of tyrosinase activity to adrenergic innervation. Nature 255. 152-3. Leschey, K. H.. Hackett, S. F., Singer, J. H. and Campochiaro. P. A. (1990). Growth factor responsiveness of human retinal pigment epithelial cells. Invest. OphthaZmoZ. Vis. Sci. 31, 839-46. McQuillan, A., Hutchinson, M. and Panasci, 1.. C. (1989). Evidence for increased aMSH receptor binding in the F, variant of B,, melanoma cells grown in dialyzed fetal calf serum. 1. Cell Physiol. 141, 281-3. Miller, S. S. and Steinberg, R. H. (19 77a). Active transport of ions across frog retinal pigment epithelium. Exp. Eye Res. 25, 235-48. Miller. S. S. and Steinberg, R. H. (1977b). Passive ionic properties of frog retinal pigment epithelium. 1. Membr. Biol. 36, 337-72. Pfeffer. B. A.. Clark, V. M.. Flannery. J. G. and Bok. 1). ( 1986). Membrane receptors for retinol-binding protein in cultured human retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 27, 103140. Slominski. A., Moellmann, G., Kuklinska, E., Bomirski, A. and Pawelek, J. (1988). Positive regulation of melanin pigmentation by two key substrates of the melanogenic pathway, L-tyrosine and r.-dopa. 1. Cell Sci. 89. 28 7-96. Steinberg. R. H., Miller, S. S. and Stern, W. H. ( 19 78). Initial observations on the isolated retinal pigment epitheliumchoroid of the cat. Invest. Ophthalmol. Vis. Sci. 17. 675-8.

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Influence of bicarbonate and insulin on pigment synthesis by cultured adult human retinal pigment epithelial cells.

Exp. Eye Res. (1991) 53, 681-684 LETTER TO THE EDITORS Influence of Bicarbonate and Insulin on Pigment Synthesis Cultured Adult Human Retinal Pigm...
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