Biomedical subjects

D L Epstein

Publications and source records attributed to D L Epstein.

At least 37 records · Page 2Linked to original sources

PubMed1999

Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions.

The regulation of transendothelial fluid flow by glucocorticoids was studied in vitro with use of human endothelial cells cultured from Schlemm's canal (SCE) and the trabecular meshwork (TM) in conjunction with computer-linked flowmeters. After 2-7 wk of 500 nM dexamethasone (Dex) treatment, the following physiological, morphometric, and biochemical alterations were observed: a 3- to 5-fold increase in fluid flow resistance, a 2-fold increase in the representation of tight junctions, a 10- to 30-fold reduction in the mean area occupied by interendothelial "gaps" or preferential flow channels, and a 3- to 5-fold increase in the expression of the junction-associated protein ZO-1. The more resistive SCE cells expressed two isoforms of ZO-1; TM cells expressed only one. To investigate the role of ZO-1 in the aforementioned Dex effects, its expression was inhibited using antisense phosphorothioate oligonucleotides, and the response was compared with that observed with the use of sense and nonsense phosphorothioate oligonucleotides. Inhibition of ZO-1 expression abolished the Dex-induced increase in resistance and the accompanying alterations in cell junctions and gaps. These results support the hypothesis that intercellular junctions are necessary for the development and maintenance of transendothelial flow resistance in cultured SCE and TM cells and are likely involved in the mechanism of increased resistance associated with glucocorticoid exposure.

Body Fluids↗

PubMed1999

Identification and isolation of differentially expressed genes from very small tissue samples.

Identification of differentially expressed genes from tissue samples weighing only a few milligrams has remained a major challenge. Here, we describe a novel and simple strategy that uses standard molecular biology equipment and commercially available kits. The approach combines isolation of total RNA by silica-gel binding, reverse transcription using anchored modified, 5' end enhancers oligonucleotides, exponential amplification of the single-stranded cDNA and hybridization to high-density cDNA filter arrays. The method was tested by comparing genes expressed on freshly isolated human trabecular meshwork tissue with those expressed in corresponding primary cells at third passage. Validation was achieved by using two biological properties: (i) hybridization, to identify the differentially expressed genes, and (ii) PCR amplification, to confirm their distinct expression. The strategy presented allows the identification of differentially expressed genes and/or uncharacterized expressed sequence tags (ESTs) in very small tissue samples, including those from clinical specimens.

Biotechnology↗

PubMed1999

Hydraulic pressure stimulates adenosine 3',5'-cyclic monophosphate accumulation in endothelial cells from Schlemm's canal.

PURPOSE: Fluid flow across various endothelia results in a variety of intracellular and extracellular adaptations. In the living eye, aqueous humor flows across the surface of endothelial cells on trabecular meshwork (TM) beams and in the juxtacanalicular tissue and through or between a continuous monolayer of endothelial cells that line Schlemm's canal (SC). This study was undertaken to test the hypothesis that fluid flow induces biochemical changes in the endothelial cells of the outflow pathway that may modify outflow resistance. METHODS: Trabecular meshwork and SC cells isolated from the outflow pathway of human cadaveric eyes were seeded onto porous filters, placed in Ussing-type chambers, and subjected to fluid flow driven by a pressure head of 15 mm Hg on their apical surface. Cell lysates were prepared and analyzed for adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Barrier function of cell monolayers was examined using transendothelial electrical resistance measurements. RESULTS: Three different SC cell strains in 14 independent experiments responded with at least a threefold increase in cAMP that was both time and pressure dependent. Conversely, flow-treated TM cells failed to respond in six independent experiments in which five different TM cell strains were used. Electrical resistance across cell monolayers positively correlated with cAMP accumulation and was calcium sensitive. CONCLUSIONS: cAMP signaling is affected by pressure differentials across SC cell monolayers and provides evidence for the participation of SC cells in the regulation of aqueous outflow.

Anterior Eye Segment↗

PubMed1999

Modulation of myocilin/TIGR expression in human trabecular meshwork.

PURPOSE: To study factors that modulate myocilin/trabecular meshwork inducible glucocorticoid response protein (TIGR) mRNA expression in human trabecular meshwork (TM). METHODS: mRNA from fresh TM of four human donors, from perfused anterior segment organ cultured TM of three donors, and from four primary TM cell lines of different donors was isolated. The full length cDNA of myocilin/TIGR was cloned from TM mRNA using a polymerase chain reaction approach and used as probe for northern blot analysis hybridization. Trabecular meshwork cell cultures were treated with transforming growth factor (TGF)-beta1 (1 ng/ml), dexamethasone (10(-7) M), and mechanical stretch (10%). RESULTS: mRNA for myocilin/TIGR could be readily detected by northern blot analysis hybridization in 2 to 3 microg of total RNA from all fresh and all organ-cultured TM samples. In contrast, no mRNA for myocilin/TIGR could be detected in 20 microg of total RNA isolated from three different primary TM cell lines. Only one TM cell line had a baseline expression of myocilin/TIGR, which was 35- to 55-fold lower than that of fresh or organ-cultured TM samples. Treatment of TM cell cultures with dexamethasone for 1 day markedly increased expression of myocilin/TIGR mRNA, an effect that was even more pronounced after 3 days of treatment. Treatment with TGF-beta1 for 24 hours had no effect; however, after 3 and 12 days of treatment a 3.8- and 4-fold increase in myocilin/TIGR mRNA expression was observed. Expression of myocilin/TIGR mRNA was also increased after 10% mechanical stretch; however, in contrast to the effects of TGF-beta-1, this effect was observed much earlier (8-24 hours) after treatment. CONCLUSIONS: Dynamic mechanical stimuli maintain myocilin/TIGR expression in TM in situ and lack of these stimuli in monolayer cell cultures might be involved in downregulation of myocilin/TIGR expression.

Adult↗

PubMed1999

Acto-myosin drug effects and aqueous outflow function.

PURPOSE: Previous studies have identified the cytoskeletal proteins actin and tubulin as potential cellular targets in the trabecular meshwork for novel glaucoma therapy. The authors and others have hypothesized that acto-myosin interactions may be important for outflow function. The current study was conducted to evaluate 2,3-butanedione 2-monoxime (BDM), a compound that interferes with acto-myosin function through the myosin adenosine triphosphatase (ATPase) reaction; 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a proposed myosin light-chain kinase inhibitor; and the direct actin disrupter, latrunculin B, in an outflow pathway cell culture and perfused excised eye model system. METHODS: Freshly enucleated porcine eyes were perfused using the constant-pressure method at 15 mm Hg and 25 degrees C. Human trabecular meshwork (HTM) cells and Schlemm's canal (SC) cells were grown in culture, treated with BDM, H-7, and latrunculin B, and then fixed, stained for beta-tubulin and filamentous actin, and observed by epifluorescence. RESULTS: Twenty millimolar BDM, 100 microM H-7, and 1 microM latrunculin B increased outflow facility 36%, 63%, and 72%, respectively, compared to sham-treated controls, 13%, 15%, and 4% (n=7, 8, and 8; P=0.01, 0.0001, and 0.0002), respectively. In cultured HTM and SC cells, 100 microM H-7 caused a rapid loss of filamentous actin staining but did not produce a change in cell shape or cell- cell attachment. In contrast, 20 mM BDM induced a loss of cell- cell attachment and a change in cell shape that was associated with a 50% to 60% loss of filamentous actin staining, often in a distinct stick-and-ball pattern. Latrunculin B caused a severe loss of actin staining and cell shape changes. No drug altered beta-tubulin staining. CONCLUSIONS: Interference with myosin function can cause a secondary loss of actin organizational structure. Our study indicates that myosin, perhaps through its various phosphorylation reactions, may have a potential regulatory role in outflow function.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Tyrphostins disrupt stress fibers and cellular attachments in endothelial monolayers.

Endothelial permeability, which plays a critical role in many physiologic and pathologic processes, depends on the integrity of intercellular and cell-substrate attachments and the actin cytoskeleton. The proteins located at the cytoplasmic face of adherens and focal contact junctions are rich in sites of tyrosine phosphorylation. To better understand the role of tyrosine phosphorylation in regulating endothelial cell shape, actin stress fibers, and cell junctions, we treated confluent calf pulmonary artery endothelial cells with 14 different tyrphostins, a class of specific tyrosine kinase inhibitors. Using immunofluorescence microscopy to assess cell shape, phosphotyrosine levels, actin stress fibers, and focal contact and junctional proteins, we found that the effects of the tyrphostins could be grouped into three categories. Four tyrphostins had no discernible effect on stress fibers or cell attachments. Seven tyrphostins produced cell retraction with concomitant disruption of both stress fibers and cell-substrate attachments. One member of this group, tyrphostin 25, showed greater specificity for cell-cell junctions than the others, causing cell separation without significantly affecting actin stress fibers or focal contacts. The third group of tyrphostins had the opposite effect, completely disrupting stress fibers and focal contacts without causing cell separation. The ability of specific tyrphostins to disrupt cell-cell or cell-substrate attachments and/or actin stress fibers implies that a certain steady-state level of tyrosine phosphorylation is necessary to maintain these structures and that there may be independent tyrosine kinase signaling pathways controlling them. Comparison of the phosphotyrosinated proteins affected by each group of tyrphostins should provide a useful new approach toward understanding the regulation of endothelial cell-cell and cell-substrate junctions.

Actins↗

PubMed1998

Microtubule disruption leads to cellular contraction in human trabecular meshwork cells.

PURPOSE: To determine whether microtubule- and actin-altering drugs, which have been shown to increase aqueous humor outflow, cause cellular contraction in human trabecular meshwork (HTM) cells. METHODS: HTM cells were plated in culture dishes containing a polymerized deformable silicone substrate. After 48 hours, the dishes were placed on an inverted microscope and treated with ethacrynic acid, colchicine, vinblastine, cytochalasin B, or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and then recorded on videotape for 15 minutes. An increase in silicone substrate wrinkle size and/or number indicated a contraction. Sham controls were used. RESULTS: Cellular contraction was observed with ethacrynic acid, colchicine, and vinblastine in the 10(-5) to 10(-4) M dosage range. Pretreatment with H-7 blocked these effects. Cytochalasin B did not produce cellular contraction. CONCLUSIONS: Microtubule disruption causes cellular contraction in HTM cells, and this effect depends on an intact actin cytoskeleton network. Contraction of trabecular meshwork cells in response to various stimuli is an attractive hypothesis for possible homeostatic mechanisms in the outflow pathway, and this may serve as a focus for novel glaucoma drug development.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Mechanical stretch alters the actin cytoskeletal network and signal transduction in human trabecular meshwork cells.

PURPOSE: Human trabecular meshwork (HTM) cells were mechanically stretched in vitro as a potential model for the distension of this tissue that can occur in vivo in response to increased pressure gradients. Cell morphology and certain components of the signal transduction pathways, including the mitogen-activated protein kinase (MAPK) and c-Jun N-terminal protein kinase (JNK) pathways, were evaluated for stretch-induced alterations. METHODS: Primary HTM cells grown in tissue culture were subjected to a mechanical stretch lasting from 10 seconds to 4 days. The actin cytoskeletal network was visualized by phalloidin staining. Proteins phosphorylated on their tyrosine residues were isolated using an immunoaffinity system and were analyzed by gel electrophoresis and immunostaining. Mitogen-activated protein kinase activity was evaluated using an in-gel assay system, and the mRNA levels of c-fos and c-jun were determined by quantitation of competitive reverse transcription-polymerase chain reaction. In addition, the amount of c-Fos protein was estimated by chemiluminescent immunoblot analysis. RESULTS: On stretching, the HTM cells elongated but regained their normal morphologic characteristics within 24 hours. Unstretched HTM cells displayed a diffuse F-actin microfilament network, whereas stretched cells exhibited complex geodesic patterns. Ten seconds after stretching began, the level of tyrosine phosphorylation on the six major phosphoproteins significantly decreased between 80% and 100%, whereas the level of paxillin tyrosine phosphorylation significantly increased 39%. Stretching caused MAPK activity and the amount of mRNA and protein of the immediate-early gene c-fos to decrease more than 60% within 2 minutes, but within 15 to 30 minutes they increased above or equivalent to normal levels. The level of c-jun mRNA was unchanged by stretching. CONCLUSIONS: In response to a mechanical stretch, major cytoskeletal alterations occur in HTM cells, which involve changes in the levels of tyrosine phosphorylation. Mechanotransduction (signal transduction by mechanical stimulation) through the MAPK signaling pathway was significantly depressed immediately after stretching; however, the JNK pathway appeared to be unaffected. The data suggest that HTM cells adapt to mechanical stress by altering the cytoskeletal network and signaling cascades.

Actin Cytoskeleton↗

PubMed1998

Gene transfer to the human trabecular meshwork by anterior segment perfusion.

PURPOSE: To determine whether adenovirus vectors are capable of transferring a foreign active protein to the perfused anterior segment of the human eye. METHODS: Primary cultures from the human trabecular meshwork tissue were exposed to replication-deficient adenovirus Av1LacZ4 carrying the reporter beta-galactosidase gene driven by the Rous Sarcoma Virus promoter. Anterior segments of six pairs of human eyes from normal donors were placed in organ culture and were perfused with culture medium at 2.5 microl/min constant flow. After 24 hours, one eye was injected once with 8 X 10(8) plaque-forming units (20 microl) of the viral vector, while the paired eye was injected with vehicle. Forty-eight hours (four pairs) and 7 days (two pairs) after injection, tissues were fixed, were assayed histochemically for transferred enzyme activity, and were analyzed morphologically. RESULTS: In monolayers, gene transfer occurs very efficiently in all distinct types of human outflow pathway cells. All human anterior segments injected with the adenovirus vector showed active gene transfer in cells of the outflow pathway: trabecular, juxtacanalicular, and inner wall of Schlemm's canal. Expression of the reporter enzyme was still present at 7 days after treatment. No activity was observed in any of the paired, vehicle-injected controls. Cell morphology and tissue architecture appeared normal in treated and control tissues, although some trabecular cell loss was observed in the corneoscleral and uveal regions of the perfused treated eyes. CONCLUSIONS: Adenoviral vectors were able to transfer active foreign genes into perfused, intact human trabecular meshwork.

Adenoviridae↗

PubMed1998

Isolation, culture, and characterization of endothelial cells from Schlemm's canal.

PURPOSE: An important goal in glaucoma research has been to understand the functional contribution of trabecular meshwork (TM) and Schlemm's canal (SC) endothelia to aqueous humor outflow resistance. To date, TM cells are routinely cultured and used as a model by several laboratories. However, there has been only limited success in isolating SC cells. The current objective was to develop a technique for selective isolation and culture of endothelial cells from human SC. METHODS: The anterior chamber of human cadaveric eyes was cut into eight equal and radially symmetric wedge-shaped pieces. Using a dissecting microscope, a gelatin-coated suture (6-0 sterile nylon monofilament) was gently inserted into the lumen of SC and advanced into the canal. The cannulated pieces of tissue were placed in culture medium and maintained for 3 weeks. Sutures were removed from SC and cells seeded onto 3-cm culture plates. Morphology, growth characteristics, and expression of endothelial surface antigens and other cellular markers were evaluated. RESULTS: Of the 20 pairs of eyes that were cannulated, primary cells were obtained from 13. All SC cell isolates had a fusiform morphology; formed nonoverlapping, linearly arranged monolayers; and were contact inhibited. Schlemm's canal cell isolates reacted with antibodies specific for CD44 (hyaluron receptor), CD54 (intercellular adhesion molecule-1, ICAM-1), tissue-type plasminogen activator, and TM-inducible glucocorticoid-responsive protein-myocilin (TIGR-MYOC). Unlike TM cells, however, TIGR-MYOC protein was not induced in SC cells after long-term dexamethasone treatment. Schlemm's canal cells endocytosed low-density lipoprotein and acetylated low-density lipoprotein, and in the presence of Matrigel organized into multicellular tubelike structures. CONCLUSIONS: Cannulation of SC with gelatin-coated suture material is an effective method for the isolation of human SC cells and provides a cellular model to study the potential role of SC cells in aqueous humor outflow function.

Adult↗

PubMed1997

Transient loss of alphaB-crystallin: an early cellular response to mechanical stretch.

Human trabecular meshwork (HTM) is distended and stretched with increases in intraocular pressure. During this stretching, there is a rearrangement of actin filaments. The HTM cells express alpha B-crystallin, a small heat shock protein that may have a role in the stabilization and regulation of the cytoskeleton in mammalian cells. The levels of alpha B-crystallin were examined in trabecular meshwork cells after mechanical stretch. Human TM primary cell cultures, plated onto silicone sheets, were subjected to a single 10% linear stretch and samples were prepared at various times after stretch for immunoblotting or Northern blotting. Immunoblots of total protein extracts with antibody specific for alpha B-crystallin detected a 26% decrease of cellular alpha B-crystallin levels within 2 minutes. After 1 hour alpha B-crystallin levels had decreased 90% compared to control cells. The levels of alpha B-crystallin began to recover in cells stretched for 2 hours and returned to initial levels by 24 hours. Northern blots probed with alpha B-crystallin exon III cDNA detected a transcript of 0.65 kb in human TM cells and the levels of the alpha B mRNA remained constant during alpha B-crystallin protein decrease. Later, levels of the 0.65 kb transcript of alpha B-crystallin increased during the cellular recovery. These results suggest that decreased levels of alpha B-crystallin after mechanical stretch were probably not due to transcriptional changes but rather to increased degradation of alpha B-crystallin protein. An increase in mRNA levels may play a role in the recovery of alpha B-crystallin during reorganization of the cytoskeleton and attachment to the substratum. These data raise the possibility of a specific proteolysis of alpha B-crystallin protein in cells after a physiological challenge.

Blotting, Northern↗

PubMed1997

n-ethylmaleimide and ethacrynic acid inhibit kinesin binding to microtubules in a motility assay.

Treatment of proteins in vitro with sulfhydryl (SH)-reactive compounds has been used successfully to determine protein regions critical for normal function. To probe structure-function relationships in the microtubule (MT) motor kinesin, the motor was treated with two SH reactive compounds, n-ethylmaleimide and ethacrynic acid, and its function was assayed by motility and co-sedimentation techniques. In the motility assay, treatment of kinesin either before or after adsorption to the glass surfaces of a flow cell was found to inhibit the ability of coverslip-bound kinesin to bind to MTs. Inactivation of MT binding was slow, required high molar excess of the SH-reactive drug, and was very sensitive to temperature. Inhibition of MT binding occurred well after complete modification of kinesin light chain, but paralleled modification of the kinesin heavy chain. The results point to a model in which one critical cysteine per kinesin heavy chain is relatively inaccessible to solvent. Surprisingly, when the interaction between modified kinesin and MTs was examined by a co-sedimentation assay, kinesin retained the ability to bind MTs. These contrasting results may be due to conformational differences in the kinesin molecule that exist in the two assays.

Animals↗

PubMed1997

A mechanism for trabecular meshwork cell retraction: ethacrynic acid initiates the dephosphorylation of focal adhesion proteins.

Ethacrynic acid (ECA) increases aqueous humor outflow facility in human and animal model systems, and causes cellular retraction in cultured trabecular meshwork (TM) cells. ECA-induced retraction, a possible correlate to the opening of spaces in the outflow pathway in vivo, takes place coincident with disruption of cell-cell attachments and actin stress fibers. Tyrosine phosphorylated proteins are located predominantly where actin filaments terminate at sites of cell-to-cell and cell-to-substrate adhesion, and are understood to regulate cellular adhesions and filamentous (F) actin organization in many cell types. In the present study we investigated whether ECA might affect cell adhesions and F-actin in TM cells by altering levels of phosphotyrosine. We analysed levels of phosphotyrosine in cultured human TM and calf pulmonary artery endothelial cells after exposure to ECA. Using immunoflourescence microscopy and antibodies to phosphotyrosinated proteins we found a rapid decrease in phosphotyrosine levels at the focal contacts of cells treated with ECA. Immunoblots of whole cell extracts showed a decrease in phosphotyrosine predominantly in a band running at about 120 kD, with a more subtle decrease in a band about 65 kD. Reprobing the blot with antibodies to pp120 focal adhesion kinase (FAK) or paxillin indicated that the 120 kD band was FAK and the 65 kD band was likely paxillin. Immunoprecipitation of FAK or paxillin and probing the resulting blot with antibodies to phosphotyrosine confirmed that these proteins were rapidly dephosphorylated after ECA addition. Loss of FAK and paxillin proteins in cells was then confirmed using immunofluorescence microscopy. Dephosphorylation of these proteins was detected before the onset of retraction, stress fiber disruption, or complete disruption of focal adhesions. A pure microtubule inhibitor (colchicine), did not cause stress fiber disruption or decrease focal adhesion phosphorylation. We postulate that dephosphorylation of FAK and paxillin by ECA disrupts signaling pathways that normally maintain the stability of the actin cytoskeleton and cellular adhesions, and that this action leads both to cell shape change in culture, and to facility changes in vivo.

Actins↗

PubMed1997

Nonsulfhydryl-reactive phenoxyacetic acids increase aqueous humor outflow facility.

PURPOSE: The phenoxyacetic acid, ethacrynic acid (ECA), has potential use in glaucoma therapy because it acts to increase aqueous outflow in vivo and in vitro. In human trabecular meshwork (HTM) cell culture, ECA acts to change cell shape and attachment, effects that have been correlated with microtubule (MT) alterations and chemical sulfhydryl (SH) reactivity. To further explore these actions, we evaluated two non-SH reactive phenoxyacetic acids, inadcrinone and ticrynafen, and the MT-disrupting drug vinblastine. METHODS: Excised bovine and porcine eyes were perfused and outflow facility measured. Calf pulmonary artery endothelial and HTM cells were grown in culture and cytoskeletal effects evaluated after drug treatment. RESULTS: Indacrinone, ticrynafen, and vinblastine all caused an increase in outflow facility. In contrast with ECA, the outflow effects of indacrinone and ticrynafen were not blocked by excess cysteine. Although indacrinone and ticrynafen produced changes in cell shape in vitro, the beta-tubulin staining pattern of treated cells was not altered. Vinblastine caused cell shape change and the expected MT disruption. CONCLUSIONS: Phenoxyacetic acids can increase aqueous outflow facility and alter HTM cell shape and attachment in vitro by a non-SH, non-MT mechanism (which is probably shared also by ECA). These findings suggest the possibility of a broader class of glaucoma drugs that may be directed at the HTM. An understanding of the cellular target for these drugs has implications both for potential glaucoma therapy and for the cytoskeletal mechanisms involved in normal outflow function.

Animals↗

PubMed1997

Laser trabeculoplasty: how little is enough?

BACKGROUND AND OBJECTIVE: The optimal number of laser applications for argon laser trabeculoplasty in patients with primary open-angle glaucoma has not been established. The purpose of this study was to determine the fewest number of burns necessary for clinically effective intraocular pressure reduction for these patients. PATIENTS AND METHODS: Using a prospective, randomized, collaborative study, the authors examined the relationship between the number of laser trabeculoplasty burns and intraocular pressure for patients with primary open-angle glaucoma. A total of 122 patients received either 50 burns to half of the trabecular meshwork (group 1) or 35 burns to one third of the trabecular meshwork (group 2). RESULTS: The mean baseline intraocular pressures were similar between group 1 (22.3 +/- 4.1 mm Hg [mean +/- SD]) and group 2 (22.8 +/- 5.0 mm Hg) (P = .58). Intraocular pressure reduction at 9 to 12 months, although significant in both groups (group 1: 4.4 +/- 3.0 mm Hg, P < .0001; group 2: 3.9 +/- 5.1 mm Hg, P < .0001), did not differ significantly between the two groups (P = .63). CONCLUSION: This study demonstrated that 35 burns may be as clinically effective as 50 burns in reducing the intraocular pressure in primary open-angle glaucoma patients.

Aged↗

PubMed1996

Characterization of alpha 2-macroglobulin binding to human trabecular meshwork cells: presence of the alpha 2-macroglobulin signaling receptor.

Direct binding of receptor-recognized alpha 2-macroglobulin (alpha 2M*) or a cloned receptor binding fragment from rat alpha 1-macroglobulin (RBF) to human trabecular meshwork cells demonstrated two classes of cell surface binding sites. One class has an apparent Kd of 5.0 nM and a receptor number of 31,800 receptors/cell. The other class has an apparent Kd of 20 pM and a receptor number of 1600 receptors/cell. Binding studies of alpha 2M* or RBF in the presence of a competitor for binding to low-density-lipoprotein receptor-related protein/alpha 2M* receptor (LRP/alpha 2MR) called receptor-associated protein (RAP) show that only the lower affinity class of binding sites is susceptible to competition with RAP. Uptake studies demonstrate specific internalization and degradation of alpha 2M* which is inhibitable by RAP. Exposure of the cells to alpha 2M* and RBF (40 nM) is associated with mean increases of 171 and 210%, respectively, in the intracellular calcium concentration, which is not inhibitable by RAP or pertussis toxin. These studies present the first characterization of alpha 2M* and RBF signaling in a primary human cell type and suggest a role for alpha 2M* in the physiology of the eye.

Aged↗

PubMed1996

Dexamethasone inhibits trabecular cell retraction.

Glucocorticosteroids such as dexamethasone (Dex) are known to cause an increased resistance to aqueous outflow in the intact and cultured eye. We investigated whether Dex treatment of cultured endothelial or trabecular meshwork (TM) cells might interfere with the cell separations and retraction induced by the facility-enhancing agents ethacrynic acid (ECA), cytochalasin B and the calcium chelator EGTA. Our hypothesis was that Dex-induced changes in the response of our model cells in vitro might serve as a paradigm for those produced in the cells of the outflow pathway, perhaps through influencing the changing dimensions of the pathway for aqueous humor through the juxtacanalicular tissue and/or inner wall of Schlemm's canal. We treated calf pulmonary artery endothelial (CPAE) and human and porcine TM cells with Dex (1-100 microns, 1-9 days), and then assessed monolayer and cytoskeletal integrity by immunofluorescence microscopy for tubulin and direct fluorescence staining for F-actin after exposure to the agents named above. We found that Dex-pretreated CPAE and TM cells gradually (over 5-7 days) became refractory to the effects of both ECA and EGTA, but not to cytochalasin B. Despite the preservation of general cell shape and attachment after ECA in Dex-treated cells, microtubule disruption still took place as in controls. Dex-treated cells also demonstrated a reorganization of filamentous actin staining after ECA and EGTA. Combination experiments of ECA and EGTA in Dex-treated cells suggested that the Dex effects were due to a greater strength of cell-to-cell and cell-to-substrate attachment, possibly due to interference with the normal cellular signaling required for coordinated cellular retraction and junctional disruption.

Actins↗

PubMed1996

Drug-induced transient myopia and angle-closure glaucoma associated with supraciliary choroidal effusion.

PURPOSE: We investigated the mechanism of drug-induced transient myopia, anterior chamber shallowing, and secondary angle-closure glaucoma in a young woman. METHODS: Ultrasound biomicroscopy was performed and the effects of cycloplegic eyedrops and unilateral laser iridotomy were evaluated. RESULTS: Cycloplegic eyedrops and unilateral laser iridotomy had no effect. Ultrasound biomicroscopy identified the presence of a supraciliary choroidal effusion that caused forward displacement of the lens-iris diaphragm, resulting in increased myopia, anterior chamber shallowing, and angle-closure glaucoma. Discontinuance of trimethoprim and sulfamethoxazole combination led to the complete resolution of the condition. CONCLUSIONS: Idiosyncratic drug reactions may produce a supraciliary choroidal effusion, resulting in myopia and secondary angle-closure glaucoma from the induced forward shift in the position of the crystalline lens and ciliary body.

Adult↗