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The purpose of the present study was to develop methods for isolation, purification and cultivation of human conjunctival melanocytes. Conjunctiva excised from donor eyes or corneal rims was subjected with various enzyme digestion methods or by the enzyme-microdissection method. Cells were cultured with F12 medium supplemented by fetal bovine serum, basic fibroblast growth factor, isobutylmethylxanthine and cholera toxin. Contaminant cells were eliminated by a selective cytotoxic agent, geneticin. Both trypsin digestion and dispase-microdissection methods provided pure conjunctival melanocyte cultures with high cell yields, good viability and rapid growth rate. Melanocytes isolated with dispase-microdissection method showed better viability and growth capacity. Cells grew well, could be passaged for 5-10 generations and divided 20 times in vitro. They maintained a constant melanin content per cell and produced measurable amounts of melanin in vitro. Melanogenesis correlated with the degree of pigmentation of the eyes (iris color). This method provides a valuable source of large numbers of human conjunctival melanocytes, which can be used to study their biological behavior, to compare with the epidermal and uveal melanocytes; and to compare them to their malignant counterparts in the exploration of the pathogenesis of conjunctival melanoma.

The effects of various prostanoids on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (bFGF) (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium or various deleted media with or without various prostanoids at different concentrations. After 6 days, the numbers of cells and dendrites were counted and melanin content was measured and compared with controls. Prostaglandin E(2), an EP(2)receptor agonist (AH 13205) and AGN 192093 (thromboxane mimetic) stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. A mixed EP(1)and EP(3)receptor agonist (sulprostone), a EP(4)receptor agonist (ONO-AE1-329), IP receptor agonists (cicaprost or iloprost) and a TP receptor agonist (U-46619) showed no effect. Prostaglandin D(2)showed stimulating effects. However, these stimulating effects could not be blocked by the addition of a DP receptor antagonist (BW A868C). Furthermore, a DP receptor agonist (BW 245C) showed no effects, indicating that the effect of prostaglandin D(2)may involve receptors other than the DP receptor subtype. The present study indicates that: (1) among various EP receptor agonists, only an EP(2)receptor agonist has stimulating effects on iridal melanocytes; (2) DP, IP and TP receptor agonists do not have stimulating effects; and (3) the mechanisms of action of prostaglandin D(2)and AGN 192093 need further study., (Copyright 2001 Academic Press.)

The influence of autonomic neurotransmitters on the growth and melanogenesis of cultured uveal melanocytes was studied. Uveal melanocytes were cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (complete medium). The cells were plated into multiple well plates, and various concentrations of adrenergic and cholinergic agents were added to the media (complete medium or various deleted media). After 6 days, the cells were detached for cell counting and melanin measurement and compared to controls. Epinephrine, isoproterenol, salbutamol and metaproterenol (adrenergic agonists that can activate beta(2)-adrenoceptors) substantially stimulated growth and melanogenesis of cultured uveal melanocytes in cAMP-deleted medium. Methoxamine, clonidine, prenalterol and D-7114 (adrenergic agonists that do not activate beta(2)-adrenoceptors) showed no effect under similar experimental conditions. Muscarine (a cholinergic agonist) inhibited the growth and melanogenesis of uveal melanocytes in complete medium. It indicates that adrenergic agents (beta(2)-adrenoceptor agonists) stimulate growth and melanogenesis in uveal melanocytes, while cholinergic agonist has an inhibitory effect. This effect appears to involve the cAMP second messenger system. These studies suggest that homeostasis of the uveal melanocytes may be maintained, in part, by regulating the autonomic nervous system in vivo., (Copyright 2000 Academic Press.)

The effect of various prostaglandins on the growth, melanogenesis and dendrification of cultured iridal melanocytes were studied. Iridal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor (complete medium). The iridal melanocytes were plated into multiple well plates and cultured with complete medium, cAMP-deleted medium with or without various prostaglandins at different concentrations. After 5-7 days, the numbers of cells and dendrites were counted and melanin content was measured and compared to controls. Prostaglandin A(2)and E(1)stimulated growth, melanogenesis and dendrification of cultured iridal melanocytes in cAMP-deleted medium. Prostaglandin F(2 alpha), PhXA85 and latanoprost showed no effect under similar experimental conditions. These results indicate that prostaglandin A(2)and E(1)stimulate growth and differentiation of iridal melanocytes through the activation of cAMP system. Failure of stimulation of melanogenesis of iridal melanocytes by prostaglandin F(2 alpha)and PhXA85 in vitro may be related to a different second messenger system activated by these prostaglandins and the selective stimulating effect of these prostaglandins on iridal melanocytes from mixed-colored irides., (Copyright 2000 Academic Press.)

The presence of eumelanin and pheomelanin in irides from eyes of various colors was determined and quantified by a highly specific microanalytical procedure based on chemical degradation. Significant differences in the type of melanin were observed in the stroma and iris pigment epithelium (IPE) fractions obtained by micro-dissection of the iris specimens. Melanin from the IPE is essentially eumelanin, while the pigment in IPE-scraped iris (consisting mainly of stroma plus anterior IPE) proved to be both eumelanic and pheomelanic. A pheomelanic-type pigmentation was associated with green irides, while green-blue mixed-color irides were mostly eumelanic; by contrast, green-brown mixed-color and brown irides could not be placed into either of the two categories and probably feature a mixed pigment content. Blue irides invariably exhibited very low pigment content. Analysis of cultured iridial melanocytes in the growing stage showed a significant shift to pheomelanic pigmentation when compared with those in IPE-scraped tissues, providing evidence that growth of iridial melanocyte induce a marked change of melanin metabolism. After senescence, cultured melanocytes exhibited a marked increase in pigment content, most of the variation was associated with the eumelanin content. These results represent the first direct evidence for the presence of eumelanin and pheomelanin in human irides, and suggest that differences in stromal pigmentation are due not only to the quantity, but also the nature of the melanin pigment., (Copyright 1998 Academic Press.)

The effect of TGF-beta2 on growth of uveal melanocytes in vitro was studied and the dose-dependent inhibitory effect of TGF-beta2 was compared with the known concentration of TGF-beta2 in aqueous humor. Uveal melanocytes were isolated and cultured with medium supplemented with cAMP elevating agents and basic fibroblast growth factor. The uveal melanocytes were plated into multi-well plates. After 24 hr, TGF-beta2 was added to the medium in various concentrations. After 5 days, the cells were detached, counted and compared to the controls. The effect of TGF-beta2 on DNA synthesis (as evaluated by uptake of bromodeoxyuridine) were also tested. TGF-beta2 inhibited growth and DNA synthesis of cultured uveal melanocytes in a dose-dependent manner at concentrations from 0.03-10.0 ng ml-1. The growth-inhibition of TGF-beta2 was present even in serum-free medium. TGF-beta2 had little or no effect on melanogenesis of cultured uveal melanocytes. The serum used for cultivation did not contain active TGF-beta1 or TGF-beta2 as measured by immunoassay. The known amount of active TGF-beta2 in aqueous humor (0.2-0.4 ng ml-1) is sufficient to inhibit the growth of uveal melanocytes. It indicates that TGF-beta2 is a potent growth inhibit factor of uveal melanocytes and may play an important role in maintaining the non-proliferative, relatively quiescence status of uveal melanocytes in vivo., (Copyright 1998 Academic Press)

The purpose of this study was to investigate factors regulating melanogenesis in cultured human uveal melanocytes. The effects of various substances on the melanin content, tyrosinase activity and growth of cultured uveal melanocytes were tested. 12-O-tetradecanoyl-phorbol-13-acetate (a protein kinase C activator) and various cAMP-elevating agents, including isobutylmethylxanthine, cholera, toxin, and dibutyryl-cAMP increased melanin content per culture, tyrosinase activity and cell numbers of uveal melanocytes in a dose dependent manner. Basic fibroblast growth factor (tyrosine kinase activator) stimulated growth but did not affect melanin content per culture of uveal melanocytes in vitro. These results indicate that cAMP-elevating agents and protein kinase C activator stimulate melanogenesis and growth of cultured uveal melanocytes. Tyrosine kinase activator stimulates growth but not melanogenesis of cultured uveal melanocytes.