Potentiation of glucocorticoid activity by 5 beta-dihydrocortisol: its role in glaucoma.

5 beta-Dihydrocortisol potentiated the threshold level (the smallest dose producing a measurable effect) of topically applied cortisol (0.02 percent) and dexamethasone (0.003 percent) in causing nuclear translocation of the cytosolic glucocorticoid receptor in rabbit iris-ciliary body tissue. 5 beta-Dihydrocortisol accumulates in cells cultured from trabecular meshwork specimens from patients with primary open-angle glaucoma, but not in similar cells derived from nonglaucomatous patients. In view of the sensitivity of patients with primary open-angle glaucoma to the effects of glucocorticoids in raising intraocular pressure, this potentiation may be responsible for the steroid sensitivity and for the ocular hypertension seen in this disorder.

Receptors for glucocorticoids in the lens epithelium of the calf.

The calf lens epithelium contains a specific cytoplasmic receptor for glucocorticoids. This binding protein has a high affinity for dexamethasone (average dissociation constant, 8 x 10(-9) mole per liter), a low capacity (average, 550 femtomoles per milligram of protein), extreme heat sensitivity, and exhibits a pattern of competition similar to that of glucocorticoid receptors in other tissues. This provides direct biochemical evidence that these tissues may function as a target organ for glucocorticoids.

Ectopic expression of a mutant form of PKCalpha originally found in human tumors: aberrant subcellular translocation and effects on growth control.

A point mutation in PKCalpha was originally discovered in a subpopulation of human pituitary tumors characterized by their invasive phenotype, and the same mutation was also seen in some thyroid neoplasms. To investigate the role of this mutation in tumorigenesis, normal and mutant human PKCalpha cDNAs were overexpressed in Rat6 embryo fibroblasts (R6). When extracts of R6 cells that expressed either the normal or mutant PKCalpha were assayed in the presence of calcium, phosphatidylserine and the phorbol ester TPA, for phosphorylation of either histone IIIS or the EGF-receptor peptide, both extracts gave similar results. However, the subcellular localization of the two proteins differed. Immunohistochemistry studies indicated that after treatment with TPA normal PKCalpha mainly translocated to the plasma membrane, but mutant PKCalpha translocated mainly to the perinuclear region and slightly to the nucleus. Furthermore, the cells that expressed the mutant PKCalpha displayed a decreased requirement for serum when compared to the cells expressing the normal human PKCalpha, and they formed small colonies in soft agar. By contrast, the cells expressing the normal human PKCalpha failed to form colonies in soft-agar. Thus, ectopic expression in rat fibroblasts of this mutant human PKCalpha sequence alters the growth properties of these cells and, when activated, the mutant PKCalpha displays aberrant intracellular translocation. Therefore, this mutation in PKCalpha could contribute to the process of tumor progression in certain human tumors.

Defects in cortisol-metabolizing enzymes in primary open-angle glaucoma.

Assays of cortisol-metabolizing enzymes in homogenates of human trabecular meshwork cells under optimal conditions revealed two defects in primary open-angle glaucoma (POAG): one is a marked increase in delta 4-reductase and the other is a decrease in 3-oxidoreductase. Experiments indicated that the differences in enzyme activities seen between POAG and nonPOAG trabecular meshwork derived cell homogenates were due to altered amounts of enzymes rather than to alterations in cofactor availability, pH, or endogenous activators or inhibitors. This clearly demonstrates an enzymatic defect(s) in POAG which may be the basis for the ocular hypertension and sensitivity to exogenous glucocorticoids seen in this disorder.

Arachidonic acid metabolism by cultured bovine corneal endothelial cells.

Pathways of arachidonic acid metabolism were identified in freshly prepared and in cultured bovine corneal endothelial cells. The principal pathway of arachidonic acid metabolism in the bovine corneal endothelial cells appears to be the cyclooxygenase pathway with the resultant synthesis of PGI2, PGF2 alpha and PGE2. At least two of these products, PGI2 and PGF2 alpha, are formed by the enzymatic conversion of the substrate, PGH2. Measurements of endogenous prostaglandin production by radioimmunoassay demonstrated that PGE2 was the major arachidonic acid metabolite released, with smaller amounts of PGF2 alpha and the stable hydrolysis product of PGI2, 6-keto PGF1 alpha. The release of all three prostanoids was significantly increased by the addition of the calcium ionophore (A23187), human thrombin, bradykinin and histamine. Basal and stimulated release of prostaglandins by the corneal endothelium may contribute to the regulation of intraocular pressure and also in the modulation of the corneal response to injury.

Nuclear translocation of the cytoplasmic glucocorticoid receptor in the iris--ciliary body of the rabbit.

The cytoplasmic glucocorticoid receptor of the iris--ciliary body of the rabbit has been shown to translocate to the cell nucleus within 30 min of an injection of cortisol. Over the next 2 1/2 hr the amount of receptor returns to the control value. The threshold of the loss of receptor from the cytosol was found at 0.04 mg of cortisol per kilogram body weight, with a maximal loss being reached at a cortisol dose of 0.5 mg/kg B.W. The inactive glucocorticoid tetrahydrocortisol and the major sex steroids, dihydrotestosterone, estradiol, and progesterone, had little or no effect on this translocation, indicating the specificity of the cortisol effect. Thus this receptor appears to migrate to the cell nucleus in a manner similar to that found in other steroid-sensitive tissues and is consistent with the accepted mechanism whereby these hormones regulate differential gene expression in the nucleus.

The effect of dexamethasone on the synthesis of collagen in normal human trabecular meshwork explants.

This study demonstrates that the trabecular meshwork cells of the human eye incorporate 3H-Proline into collagen during in vitro incubation. Addition of dexamethasone to the incubation mixture produced a marked decrease in this incorporation. Dexamethasone was active at 10(-8) M and higher concentrations. The specificity of the hormone effect was demonstrated by its inability to alter 3H-leucine incorporation in these cells. These results indicate that dexamethasone decreases the synthesis of collagen, a major component of the extracellular matrix, in the trabecular meshwork.

Specific glucocorticoid receptor in the iris--ciliary body of the rabbit.

The cytoplasm of the iris--ciliary body of the rabbit contains a receptor capable of specifically binding dexamethasone. This binding protein has a high affinity for dexamethasone (average KD = 2.0 X 10(-8) M), a low capacity (average 4.8 X 10(-13) mol of steroid bound per milligram of protein), and extreme heat sensitivity; it exhibits a pattern of competition virtually identical to that obtained with glucocorticoid receptors from other tissues and shows characteristic physicochemical behavior in various salt concentrations. The demonstration of a specific dexamethasone receptor in the iris--ciliary body provides the first direct biochemical evidence that these tissues may function as a target organ for glucocorticoids.

Human trabecular meshwork cells in culture: morphology and extracellular matrix components.

This study demonstrates the presence of laminin and collagen type IV in the extracellular space of human trabecular meshwork (HTM) cells in culture and its absence in cultures of fibroblasts from sclera adjacent to the outflow pathway. These basement membrane components can be detected by standard immunohistochemical techniques. Positive staining for these macromolecules is found only after the HTM cells have reached confluence. The presence of laminin and human collagen type IV in the early passages can serve as additional criteria for identification of HTM cells in culture. These cells provide a useful experimental system for studying the effects of drugs and other factors on the synthesis of components of the extracellular matrix.

Potentiation of collagen synthesis in explants of the rabbit eye by 5 beta-dihydrocortisol.

The biologic effect of 5 beta-dihydrocortisol on collagen synthesis was evaluated. The metabolite was found to potentiate subthreshold levels of dexamethasone in increasing 3H-proline incorporation in cells of the outflow region of the rabbit. Digestion of the tissue with highly purified collagenase indicated that the 3H-proline was incorporated into collagen type protein. This study demonstrates another biologic activity of 5 beta-dihydrocortisol, a metabolite found to accumulate in cells cultured from trabeculectomy specimens from patients with primary open angle glaucoma.

Intraocular hypotensive effect of a topically applied cortisol metabolite: 3 alpha, 5 beta-tetrahydrocortisol.

3 alpha, 5 beta-tetrahydrocortisol, previously considered an inactive metabolite of cortisol, was found to lower significantly the intraocular pressure (IOP) of rabbits made ocular hypertensive with dexamethasone alone or with threshold levels of dexamethasone plus 5 beta-dihydrocortisol. The ocular hypotensive effect appeared within 3-7 days after the metabolite was started and persisted through the duration of the experiments. The metabolite did not lower the IOP of ocular normotensive untreated animals. Thus, 3 alpha,5 beta-tetrahydrocortisol is a naturally occurring steroid antagonist, which may be of use in the treatment of primary open-angle glaucoma.

Dexamethasone induces specific proteins in human trabecular meshwork cells.

Previous studies have demonstrated the presence of high-affinity, glucocorticoid-specific receptors in explants of human outflow tissue and in cultured trabecular meshwork. Glucocorticoid-induced responses of scleral fibroblasts and trabecular meshwork cells were evaluated in this study. Incubation of human trabecular meshwork (HTM) and scleral fibroblast (HS) cells with 10(-7) M dexamethasone (DEX) results in a 60% inhibition of prostaglandin production. The effects of glucocorticoid treatment on cellular and secreted proteins using [35S] methionine incorporation were evaluated. Treatment of HTM cells cultured from two normal individuals with DEX induced the expression of [35S] methionine-labelled cellular proteins of 35, 65 and 70 kD, and secreted proteins of 40, 90 and 100 kD. Under the same experimental conditions, a 70 kD molecular weight cellular protein was induced in the HS cells. There were no apparent DEX-induced alterations in HS-secreted proteins. Since a functional common response to glucocorticoid treatment in both HS and HTM cells was inhibition of prostaglandin production, the dexamethasone-induced expression of the 70 kD protein in these cells may be related to this effect. Further studies are required to elucidate specific roles of the steroid-induced proteins in the effects of glucocorticoids on HTM and HS cells.

Glucocorticoid localization by radioautography in the rabbit eye following systemic administration of 3H-dexamethasone.

Dexamethasone was localized radioautographically in the nuclei of target cells of the rabbit eye following intravenous administration of the labeled steroid. Specifically bound steroid was found in the nuclei of stromal and endothelial cells of the outflow pathway region. This suggests that the glucocorticoid-induced alteration in outflow facility may be mediated by specific effects in these target cells. Nuclear localization was also found in conjunctiva, iridial smooth muscle, choroidal stroma, retina, and sclera, suggesting a physiologic role for glucocorticoids in these tissues as well.

Glucocorticoid receptor binding in bovine lens.

The present study demonstrates glucocorticoid receptor localization in the nuclei of cells of the anterior epithelium and bow region and its absence in all other regions of the lens. Both autoradiographic and biochemical studies show that triamcinolone acetonide is inactive whereas tetrahydrocortisol (previously considered to be an inactive metabolite of cortisol) is an active competitor of dexamethasone binding to the glucocorticoid receptor in the bovine lens. The consistency of these findings by both techniques supports the validity of this observation. In addition, it indicates that autoradiography can be used for studying glucocorticoid agonist/antagonist relationships in the human lens, where only small amounts of tissue are available.

Glucocorticoid target cells in human outflow pathway: autopsy and surgical specimens.

Target cells for glucocorticoids were found in trabeculectomy specimens obtained surgically from humans with glaucoma (primary and secondary) and from nonglaucomatous autopsy eyes using an autoradiographic technique. Specific nuclear localization of 3H-dexamethasone was found in cells of the trabecular meshwork, scleral fibroblasts, and in the endothelial lining of both Schlemm's canal and the outflow vessels. Glucocorticoids may alter the outflow facility by a direct effect on the metabolism of these cells. The autoradiographic method is suitable for studying competitors of glucocorticoid binding in small surgical specimens of human tissue involved in the outflow of aqueous humor.

The effect of dexamethasone on the in vitro incorporation of precursors of extracellular matrix components in the outflow pathway region of the rabbit eye.

The present study demonstrates that the cells of the outflow pathway region incorporate precursors of the extracellular matrix components during in vitro incubation. Addition of dexamethasone (10(-7) M) to the incubation mixture led to changes in precursor incorporation that were precursor and cell specific. The steroid significantly decreased the incorporation of 3H-glucosamine and increased the incorporation of 3H-proline in the outflow pathway cells and in the fibroblasts of the adjacent sclera. The specificity of the hormone effect was demonstrated by its inability to alter 3H-leucine incorporation. By contrast, in iridial fibroblasts dexamethasone caused a decreased incorporation of 3H-glucosamine while not affecting the incorporation of 3H-proline. Our results suggest that dexamethasone causes an increase in collagen synthesis in the target cells most closely related to the outflow pathway. Further, the decreased incorporation of 3H-glucosamine with dexamethasone suggests decreased synthesis of glycosaminoglycans, glycoproteins or possibly glycolipids in these same cells.

5 beta-Dihydrocortisol: possible mediator of the ocular hypertension in glaucoma.

5 beta-dihydrocortisol potentiates the action of topically applied dexamethasone in raising the intraocular pressure (IOP) in young rabbits. Dexamethasone (0.06%) plus 5 beta-dihydrocortisol (0.1 and 1.0%) elevated the IOP 7-10 mmHg within 18 days of treatment. By contrast, 0.06% dexamethasone alone raised the IOP 3 to 4 mmHg in a similar period of time. Since 5 beta-dihydrocortisol accumulates abnormally in cultured cells derived from the outflow region of the eye from patients with primary open angle glaucoma (POAG), a similar potentiation in man may account for the sensitivity of these patients to the IOP raising effect of glucocorticoids. Further, this metabolite may potentiate endogenous glucocorticoids resulting in the ocular hypertension characteristic of POAG.

Corneal-conjunctival uptake of topical 3H-dexamethasone in the rabbit eye.

Topically administered 3H-dexamethasone was localized radioautographically to the nuclei of several cell types in the cornea and conjunctiva. In the cornea, label was found in the epithelium and keratocytes. In the conjunctiva, label was found in the epithelium and in the connective tissue cells and blood vessel endothelial cells of the stroma. Prior administration of a 350-fold excess of nonlabeled hormone completely suppressed this localization, indicating that these cells are specific glucocorticoid target cells. There was no localization found in the corneal endothelium. Accumulation of silver grains between corneal stroma and Descemet's membrane suggests that this membrane acts as a partial barrier to diffusion of the steroid.

Characterization of a glucocorticoid receptor and the direct effect of dexamethasone on herpes simplex virus infection of rabbit corneal cells in culture.

Homogenates prepared from a previously established cell line derived from rabbit cornea contain a macromolecule with many properties of a glucocorticoid receptor, namely high affinity (KD = 6 x 10(-9)M) and saturable capacity (135 femtomoles/mg protein) for dexamethasone, extreme heat lability, and a pattern of competition similar to that found in other glucocorticoid target cells. Intact cells specifically bind dexamethasone with an affinity similar to that found in homogenates, and the amount of steroid bound at saturation is approximately 60,000 molecules of dexamethasone per cell. Specific dexamethasone binding was found to be localized to the cell nucleus. The corneal cells were susceptible to infection with herpes simplex virus (HSV). Dexamethasone increased cell susceptibility to the virus and facilitated the spread of the infection throughout the corneal cell culture. This effect was observed at concentrations of dexamethasone as low as 10(-9) M. Tetrahydrocortisol, an inactive glucocorticoid metabolite that does not compete with dexamethasone binding to the receptor, did not enhance HSV infection at a high concentration (10(-5) M). This study demonstrates a direct effect of dexamethasone on corneal cell-HSV interaction in the absence of exogenous immunologic factors. This effect of dexamethasone may be mediated by the glucocorticoid receptor.

Altered cortisol metabolism in cells cultured from trabecular meshwork specimens obtained from patients with primary open-angle glaucoma.

Cells cultured from trabecular meshwork specimens obtained from patients with primary open angle glaucoma (TMPOAG cells) exhibited two major differences in cortisol-metabolizing enzymes when compared with similar cells from nonglaucomatous patients. One is a marked increase (greater than 100-fold) in delta 4-reductase activity and the other is a decrease (4-fold) in 3-oxidoreductase activity. Peripheral lymphocytes from one of these patients as well as from five additional patients with POAG, did not show these abnormalities, indicating that the defects are not found in all cortisol-metabolizing cells. The abnormal metabolism of cortisol by TMPOAG cells may be of significance in the pathogenesis of POAG.