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PubMed2002

Expression of wild-type and truncated myocilins in trabecular meshwork cells: their subcellular localizations and cytotoxicities.

PURPOSE: To investigate the subcellular localizations and potential cytotoxicities of wild-type and truncated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: Full-length wild-type myocilin, truncated myocilin, and stromelysin were expressed as green fluorescence (GFP) or DsRed fusion proteins in TM cells by using adenoviral vectors, and their secretory properties and cytopathic effects were evaluated by Western blot analysis and cell proliferation assay, respectively. To determine the subcellular localizations of myocilins, the cellular organelles of the infected TM cells were stained with organelle-specific antibodies or fluorescent indicators and examined under a confocal microscope. RESULTS: Wild-type myocilin was expressed mainly in the perinuclear region of TM cells and was localized preferentially in endoplasmic reticulum (ER), but not in actin, microtubules, or mitochondria. Truncated myocilin was also localized in ER, and its expression was found to be potentially toxic to TM cells, leading to deformed cellular morphology and diminished cell proliferation, but it had no effect on the secretion of stromelysin. The truncated myocilin was also found to be colocalized with and appeared to aggregate with wild-type myocilin when the proteins were coexpressed. CONCLUSIONS: TM cells participating in the turnover of trabecular extracellular matrix (ECM) components are important in regulating aqueous outflow. The truncated myocilin, colocalized and coaggregated with wild-type myocilin, is believed to cause a dysfunction of the cells, resulting in alterations in structural compartmentalization of trabecular ECM and obstruction of aqueous outflow.

Adenoviridae↗
PubMed2005

Femtosecond laser photodisruption of human trabecular meshwork: an in vitro study.

The goal of this in vitro study was to test the feasibility of using femtosecond (fsec) laser pulses to fistulize the human trabecular meshwork (TM), and to determine the minimum exposure time and energy dosage needed to create an ablation channel. Corneo-scleral rims were obtained from tissue used for penetrating keratoplasty. Four millimeter tissue strips hydrated in Optisol-GS were used to create partial thickness fistulas in the human TM by focusing a Ti:Sapphire laser beam (45 fsec, 1 kHz, 800 nm) with various pulse energies (7.2 and 14.4 microJ) and exposure times (0.25, 0.5, 1, and 2 sec) on the inner surface of the TM. Two-photon images of the lesions were obtained with a multiphoton microscope, using an ultrafast Ti:Sapphire light source. In addition, sections of fixed tissue were examined by light microscopy. Diameters and lengths of the lesions were determined from the hematoxylin and eosin (H&E) stained sections, and the collagen structure surrounding the lesion was evaluated from the two-photon images. All selected time points (except for 0.25 sec) and energies achieved the desired photodisruption of the TM. Incisions created with 0.5 sec/14.4 microJ irradiation appeared to be the most suitable because they were able to achieve consistent full thickness trabecular ablation. Incisions created at 1 sec /14.4 microJ/pulse and 2 sec/14.4 microJ/pulse were deeper than those at shorter time points with the same pulse energy. Longer exposure times and higher pulse energies were usually more variable and associated with deeper and larger incisions and slight collateral damage. Our results indicate that, with appropriate exposure time and pulse energy, fsec photodisruption can be employed to create lesions in the human TM without damaging the surrounding tissues. This study demonstrates that fsec laser treatments may have future potential for the surgical treatment of glaucoma.

Feasibility Studies↗
PubMed2000

The similarity of protein expression in trabecular meshwork and lamina cribrosa: implications for glaucoma.

The purpose of the present investigation was to compare protein expression in various ocular cells and tissues including the human trabecular meshwork (TM) and the lamina cribrosa (LC). To conduct the comparisons, we primarily utilized autofluorography of one-dimensional (1D) and high resolution, two-dimensional (2D) polyacrylamide gels of proteins from radiolabelled tissues and cultured cells. Results from the investigations indicated that patterns of protein expression from TM and LC were the most similar among the ocular cells and tissues compared.Specifically, these autofluorographic ' fingerprints' indicated that proteins in TM and LC cultured cells and tissue were exceptionally similar (a) in band position and intensity (1D gels) and (b) in spot congruence (2D gels) as compared to other ocular cells and tissues. We conclude that the TM and the LC, two ocular tissues intimately linked to the pathogenesis of primary open-angle glaucoma, display remarkable similarity in protein expression. This finding may have implications for the molecular etiology of glaucoma.

Aged↗
PubMed2026

Lipid-nanoparticle-mediated base editing of the trabecular meshwork rescues glaucoma in vivo.

Mutations in MYOC, the most common genetic cause of glaucoma, cause misfolded myocilin to accumulate in the endoplasmic reticulum (ER), leading to trabecular meshwork (TM) dysfunction, elevated intraocular pressure, and progressive vision loss. While gene editing offers curative potential, current delivery methods rely on viral vectors, which are limited by inflammation, off-target effects, and poor translatability. Here, we report a nonviral lipid nanoparticle (LNP) platform that enables selective in vivo delivery of mRNA encoding an adenine base editor and single guide RNA (LNP-ABE) to TM cells. A direct comparison of LNP-mCherry with lentiviral GFP revealed that LNPs outperform viral vectors, achieving markedly higher efficiency and greater selectivity for the TM without inducing ocular inflammation. In a Cre-inducible Tg.CreMYOCY437H glaucoma mouse model, LNP-Cre mRNA selectively induced mutant MYOC expression in the TM, faithfully recapitulating key disease features. A single administration of LNP-ABE achieved efficient on-target editing of mutant MYOC, reducing mutant myocilin protein by approximately 46%, decreasing aggregates, alleviating ER stress, and fully rescuing the glaucomatous phenotype in Tg.CreMYOCY437H mice. Importantly, no off-target editing or ocular toxicity was detected. These findings establish LNP-based mRNA delivery as a safe, efficient, and clinically translatable approach for TM-targeted genome editing with broad therapeutic potential in glaucoma.

Animals↗
PubMed2000

Regulation of connexin phosphorylation and cell-cell coupling in trabecular meshwork cells.

PURPOSE: To investigate the expression of functional gap junctions and the effect of protein kinase C (PKC) on such junctions in confluent cultures of bovine trabecular meshwork (TM) cells. METHODS: Expression of the gap junction protein connexin43 in TM cells was examined by immunofluorescence microscopy. Intercellular communication by gap junctions was assessed by observing the diffusion of fluorescent dye from an individual cell injected with lucifer yellow. The phosphorylation of connexin43 was evaluated by immunoblot analysis with a monoclonal antibody to this protein. RESULTS: Immunofluorescence staining revealed that connexin43 was localized to sites of contact between adjacent TM cells. Exposure of cells to the PKC activator phorbol 12-myristate 13-acetate (PMA; 10 nM, 1 hour) had no marked effect on the pattern of connexin43 immunofluorescence. Injection of a TM cell with lucifer yellow resulted in the spread of the dye into neighboring cells. Dye coupling was inhibited by PMA in a dose- and time-dependent manner, and this inhibition was prevented by pretreatment of cells with the PKC inhibitor bisindolylmaleimide I. Immunoblot analysis of control TM cell lysates yielded connexin43 bands corresponding to the nonphosphorylated protein (43 kDa) and three phosphorylated forms (47, 48, and 49 kDa). Cells exposed to PMA (10 nM, 1 hour) yielded an additional band corresponding to a 44-kDa form of phosphorylated connexin43 and showed a decrease in the intensity of the band corresponding to the nonphosphorylated protein and an increase in the intensity of the 47-kDa band. CONCLUSIONS: TM cells communicate with each other through gap junctions, and the communication is inhibited by PKC, probably, at least in part, through phosphorylation of connexin43.

Actins↗
PubMed2006

Transduction of TAT fusion proteins into the human and bovine trabecular meshwork.

PURPOSE: To examine the applicability of TAT (the protein transduction domain of transactivating transcription polypeptide)-mediated protein-transduction technology, in introducing proteins of interest into trabecular meshwork (TM) cells in various culture systems. METHODS: Normal human TM cell cultures, human tissues in organ cultures, and bovine eyes in perfusion organ cultures were incubated or perfused for various lengths of time with TAT- and hemagglutinin (HA)-tagged fusion proteins, TAT-HA-beta-galactosidase (TAT-HA-beta-gal), TAT-HA-myocilin, and TAT-HA-myocilin-EGFP. Transduction of TAT-HA-beta-gal was detected by X-gal staining. Transduction of myocilin or myocilin-EGFP was evaluated by immunostaining or fluorescence. beta-Gal and EGFP proteins were used as the negative control. RESULTS: Blue X-gal staining, signifying beta-gal activity resulting from transduction, was observed in cultured TM cells in a concentration- and time-dependent manner. TAT-HA-beta-gal was also transduced into cells in all regions of TM tissues in organ cultures. TM cell cultures, after TAT-HA-myocilin incubation, showed an enhanced myocilin staining compared with the control cultures. Stronger myocilin or HA staining was also noted in TM tissues of TAT-HA-myocilin-incubated or -perfused eyes. Myocilin transduction resulted in a loss of actin stress fibers and focal adhesions in TM cells in culture. The level of phosphorylated myosin light chain was reduced. Human and bovine TM tissues after TAT-myocilin transduction also exhibited a diminished actin and paxillin-vinculin staining. CONCLUSIONS: TAT fusion proteins can be efficiently transduced into TM cells and tissues. The TAT-mediated protein transduction technology may be valuable in studies of proteins such as myocilin in the TM.

Actins↗
PubMed2005

Versican splice variants in human trabecular meshwork and ciliary muscle.

PURPOSE: Versican, chondroitin sulfate glycoprotein 2, is thought to play a role in regulating aqueous humor outflow and intraocular pressure via the human trabecular meshwork (HTM) of the eye. This protein was upregulated in HTM cells when they were treated with TGF-beta. There are four splice variant forms of versican (V0-V3) with different numbers of glycoaminoglycan (GAG) attachment domains. In this study, we investigated the various isoforms of versican from ocular tissues and cultured cells. METHODS: HTM and human ciliary muscle (HCM) tissues were dissected from three pairs of donors eyes with no histories of eye disease. Cultures of HTM and HCM cells were established from five donor eyes, and HTM cells were treated for 72 h with 1 ng/ml of either with hrTGF-beta1 or hrTGF-beta2. Total RNA was isolated from cells and tissues from each of the samples. Relative quantitation of gene expression of each variant was detected by real-time PCR with SYBR Green dye. RESULTS: All four variants of versican existed in each sample. In cultured HTM cells, the two variants with the largest number of GAG domains predominate. The V1 form is about 20% greater than the V0 form. There is an upregulation, particularly in the V0 form, when the cells are cultured. In tissue, the V1 form is about five fold greater than the other three. In the ocular ciliary muscle, the V1 form is the most prominent, but with this tissue, the relative amount of the V0 form did not change when cells were cultured. There was an upregulation of all splice variants in HTM cells when they were cultured with either TGF-beta1 or TGF-beta2. The increase in expression in the HTM with TGF-beta treatments were greatest with the V0 and V2 forms. CONCLUSIONS: This is the first report about the presence of various forms of versican in the anterior segment of the eye and the alterations in the mRNA patterns of these forms when cells are placed in culture. The results indicate the variants with the largest numbers of GAG attachment domains are the most prominent in the HTM and HCM. The increases in these forms that have the most GAG domains would be consistent with the increases in chondroitin sulfate reported in glaucoma.

Cell Culture Techniques↗
PubMed1982

Structural changes of human and monkey trabecular meshwork following in vitro cultivation.

The entire chamber angle tissue of ten monkey eyes and nine normal human eyes was cultivated in organ cultures for 1-10 days and then investigated by electron microscopy. We found that the uveal and corneoscleral trabecular cells often degenerate as early as 2-3 days after explantation, whereas the cells of the cribriform region proliferate and show an increasing number of cell organelles (mitochondria, endoplasmic reticulum and Golgi complexes). Regarding the behaviour in vitro, we distinguished three different cell populations in the trabecular meshwork with probably different functions: (1) the endothelial cells of Schlemm's canal, (2) the trabecular cells of the cribriform region and (3) the uveal or corneoscleral trabecular cells.

Animals↗
PubMed2007

Senescence in cultured trabecular meshwork cells.

BACKGROUND: It has been suggested that replicative senescence might be involved in the pathophysiology of age-related diseases. AIM: To study the process of senescence in trabecular meshwork (TM) cells. METHODS: Porcine TM tissues were obtained and placed in primary cultures with Dulbecco's modified Eagle's medium/Ham's F-12 medium. After 2-3 weeks, migrated and proliferated TM cells were trypsinised and cultured in serial passages, and identified with fluorescein-labelled low-density lipoprotein (DiI-Ac-LDL), a marker of TM cells. Staining for senescence-related beta-galactosidase activity was performed at population doubling level (PDL) 2, 8 and 16 at pH 6. Terminal restriction fragment (TRF) length was examined by Southern blot analysis using a (32)P-labelled telomere-specific sequence (TTAGGG)(3) at each PDL. RESULTS: DiI-Ac-LDL staining revealed that most (nearly 100%) of the cells in the culture were TM cells, which were flattened in shape and positive for senescence-related beta-galactosidase staining at PDL 16. Reduction of TRF length as a function of population doubling was also shown. CONCLUSIONS: TM cells exhibited characteristics of senescence at PDL 16 in vitro. The results demonstrated that cellular senescence may be related to the pathophysiology of primary open-angle glaucoma.

Animals↗
PubMed1991

Different cell populations in bovine trabecular meshwork: an ultrastructural and immunocytochemical study.

Bovine outflow tissue differs markedly from that of humans. Tissue culture studies on the cells of this region are often compared with those of primate trabecular meshwork cells. A thorough cytological and immunocytochemical characterization of the cells of the bovine chamber angle is lacking. We have therefore investigated the cells of the pectinate ligament, the reticular meshwork, the region adjacent to the aqueous plexus, the connective tissue region between reticular meshwork and ciliary muscle and the ciliary muscle itself, ultrastructurally and immunocytochemically with staining for the cytoskeletal proteins vimentin and desmin, for alpha-smooth muscle-actin and rough endoplasmic reticulum (rER). In the pectinate ligament and in the region adjacent to the aqueous plexus, the cells were found to have especially abundant rER and glycogen in their cytoplasm. Vimentin was abundant in the reticular meshwork as positive staining was seen both in frozen and paraffin sections. Alpha-smooth muscle-actin could be found in the region connecting ciliary muscle and reticular meshwork as well as in a small area adjacent to the posterior capillary loops of the aqueous plexus. Ultrastructurally, these cells resembled myofibroblasts. The ciliary muscle cells stained both for vimentin and for alpha-smooth muscle actin.

Actins↗
PubMed2003

The dual role of dexamethasone on anti-inflammation and outflow resistance demonstrated in cultured human trabecular meshwork cells.

PURPOSE: Dexamethasone (DEX) is a glucocorticoid commonly used in topical eyedrops to treat eye inflammation. It has an undesirable effect of inducing glaucoma in certain patients. In human Trabecular Meshwork (TM) cells DEX regulates a number of genes but its global influence on TM gene expression is still elusive. In the present work, DEX effects on global gene expressions of an established human TM cell line were studied by microarray. METHODS: The whole experiment of microarray was repeated three times. Differentially expressed genes were identified by an empirical Bayes approach and confirmed by Reverse Transcription Polymerase Chain Reaction. RESULTS: Eight genes (GAS1, CDH4, MT1L, CST3, ATF4, ASNS/TS11, CHOP, HSPA5) were identified that are at least a thousand times more likely to be differentially expressed due to DEX treatment and six genes (TSC22, LDHA, IGFBP2, TAGLN, SCG2, WARS) were identified that are at least a hundred times more likely to be differentially expressed due to DEX treatment. Except for MT1L, ASNS/TS11, IGFBP2, SCG2, and WARS, all the other genes are first reported here to be regulated by DEX in TM. Intriguingly, several of them have overlapping roles in anti-inflammatory response and outflow resistance. CONCLUSIONS: The results of our experiments on cultured human TM cells indicate that the increase in outflow resistance and ultimate ocular hypertension may be byproducts of the favorable anti-inflammatory response triggered by DEX.

Anti-Inflammatory Agents↗
PubMed2006

Matrix GLA protein function in human trabecular meshwork cells: inhibition of BMP2-induced calcification process.

PURPOSE: The matrix GLA (MGP) gene has been found to be among the 10 most highly expressed genes in the human trabecular meshwork (TM), and its expression is affected by conditions associated with glaucoma. Because MGP protein has been shown to play a key role in inhibiting calcification in cartilage and arterial vessels, MGP's function in human TM was investigated. METHODS: Perfused TM tissue and primary human TM (HTM) cells originated from donors of nonglaucomatous eyes. MGP mRNA was assayed by relative quantitative and real-time PCR. AdhMGP recombinant adenovirus was generated by bacterial transposition. Western blot analyses were cross-reacted with MGP N-terminal- and conformational-specific antibodies. MGP/BMP2 colocalization was analyzed by confocal microscopy. gamma-Carboxylation activity was measured by incorporation of 14CO2 into FLEEL synthetic peptide. Alkaline phosphatase (ALP) activity was used as a marker of osteogenic differentiation and a calcification precursor. Calcification was assessed by measuring direct calcium (o-cresolphthalein). Normalization was conducted with a telomerase probe (genomic DNA). RESULTS: HTM cells contained high levels of gamma-carboxylase activity and were able to convert MGP to its active conformation. Overexpression of MGP in HTM cells reduced ALP activity in a model of BMP2-induced osteogenesis. MGP colocalized intracellularly with BMP2. HTM cells aged in culture exhibited increased calcium content, increased ALP, decreased normalized MGP expression and lower gamma-carboxylase activity. CONCLUSIONS: MGP protein is active and functions as an inhibitor of BMP2-induced ALP activity in the HTM cells. The human TM may undergo a calcification process with age. Inhibition of the calcification mechanism mediated by MGP could be used to regulate resistance and elevated IOP.

Adenoviridae↗
PubMed1996

Endothelin-1 mediated Ca2+ influx does not occur through L-type voltage-dependent Ca2+ channels in cultured bovine trabecular meshwork cells.

We evaluated whether endothelin-1-induced changes in cytosolic Ca2+ concentration ([Ca2+]i) involve Ca2+ influx through L-type membrane voltage-dependent Ca2+ channels in cultured bovine trabecular meshwork (TM) cells. The cells were loaded with the fluorescent Ca2+ indicator fura-2 and cell-associated fluorescence was measured with a digital video-imaging analyzer. Application of carbachol (10(-3) M) and norepinephrine (10(-5) M) increased [Ca2+]i only transiently. Endothelin-1 (10(-9) M to 10(-7) M) also increased [Ca2+]i in a concentration-dependent manner, and its effects were larger than those of carbachol and norepinephrine. Unlike carbachol or norepinephrine, endothelin-1 evoked a peak transient effect followed by a sustained elevated level of [Ca2+]i. The only level of the sustained elevated component was dependent on extracellular Ca2+. However, pretreatment with diltiazem (10(-5) M and 10(-4) M), an L-type Ca2+ channel blocker, did not affect either component of the endothelin-1-induced increase in [Ca2+]i. These results suggest that in cultured bovine TM cells endothelin-1 receptors are coupled to Ca2+ signaling mechanisms. However, the extracellular Ca(2+)-dependent increase in [Ca2+]i may not involve Ca2+ influx through L-type Ca2+ channels.

Adrenergic alpha-Agonists↗
PubMed2006

Effect of heparin II domain of fibronectin on actin cytoskeleton and adherens junctions in human trabecular meshwork cells.

PURPOSE: To determine whether the heparin II (HepII) domain of fibronectin previously shown to increase outflow facility affects the formation and assembly of actin cytoskeleton and adherens junctions in human trabecular meshwork (HTM) cells. METHODS: Normal HTM cells and two transformed HTM cell lines were treated for 24 hours with increasing concentrations of the HepII domain. Disruptions in adherens junctions and the actin cytoskeleton were determined using immunofluorescence microscopy and Western blot analysis of immunoprecipitated cadherin/catenin complexes. Actin filaments were detected with phalloidin. Catenin (alpha and beta) and cadherin antibodies were used to detect adherens junctions. RESULTS: Treatments of cultures with the HepII domain caused cadherin/beta-catenin complexes in adherens junctions and actin filaments to disassemble. The disruption of adherens junctions and actin filaments occurred in a dose-dependent and temporal fashion. The disassembly of actin filaments occurred first, followed by the disassembly of adherens junctions. Dissociation of adherens junctions, but not actin filaments, was reversible if the HepII domain was removed. Reassembly of actin filaments required the addition of serum. Serum, however, could not trigger the reassembly of actin filaments if the HepII domain was present, suggesting that the HepII domain acted downstream of the serum stimulated RhoA activity. CONCLUSIONS: The exposure of HTM cells to the HepII domain triggers the disassembly of actin filaments and the subsequent destabilization of adherens junctions in HTM cells. This suggests that the HepII domain may increase outflow facility in cultured anterior segments by altering the organization of the TM cytoarchitecture.

Actin Cytoskeleton↗
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↗
PubMed2005

Contribution of ROCK in contraction of trabecular meshwork: proposed mechanism for regulating aqueous outflow in monkey and human eyes.

Aqueous outflow in the conventional outflow pathway is regulated by the contraction and relaxation of the ciliary muscle (CM) and the trabecular meshwork (TM). Rho-associated coiled coil-forming protein kinase (ROCK) is thought to regulate actomyosin-based contractility in many types of cells by phosphorylation of ROCK substrates. In animal models, ROCK inhibitor Y-39983 relaxed CM and TM and decreased intraocular pressure (IOP). Thus, ROCK is implicated in the regulation of aqueous outflow and IOP. However, the site of action of ROCK in monkey and man is unknown. In the present communication, RT-PCR analysis of monkey tissues showed higher levels of mRNAs for ROCK and ROCK substrates in TM compared to CM. Human TM also showed higher levels of mRNAs for ROCK and ROCK substrates compared to CM. Differences between TM and CM in human were not as high as in monkey. ROCK inhibitor Y-39983 led to a dose-dependent relaxation of carbachol-induced, contracted TM from monkey. In contrast, Y-39983 was only slightly effective in relaxing CM. Our results suggested that TM was one of the major sites for regulating IOP by ROCK. ROCK inhibitor Y-39983 might be a candidate drug for lowering IOP by increasing conventional outflow and producing fewer side effects on accommodation and miosis.

Aged↗
PubMed2006

The effect of unoprostone isopropyl on Ca2+ release-activated Ca2+ currents in cultured monkey trabecular meshwork cells and ciliary muscle cells.

PURPOSE: The aim of this study was to investigate the effect of unoprostone isopropyl (UF- 021) on Ca2+ release-activated Ca2+ (CRAC) currents in cultured monkey trabecular meshwork (TM) cells and to compare the inhibitory effects on CRAC in cultured monkey ciliary muscle (CM) cells. METHODS: Both TM and CM cells were isolated from monkey eyes, and each was grown in monolayer cell cultures. To measure changes in intracellular Ca2+ concentrations ([Ca2+]i), both types of cells were labeled with fluo-3 acetoxymethylester (AM) as a calcium indicator for 30 min at 25 degrees C and imaged with a confocal laser scanning microscope. After depletion of the intracellular Ca2+ stores with 1 microM thapsigargin and 1 mM EGTA containing Ca2+-free external solution, exposure to 2 mM Ca2+-containing external solution induced a sudden increase in [Ca2+]i, which was defined as the CRAC current. RESULTS: In both TM and CM cells, CRAC currents were observed and were well suppressed by unoprostone-free acid (M1 metabolite). In the TM cells, 3 microM M1 metabolite suppressed the CRAC current. In contrast, in the CM cells, the CRAC current was suppressed by 100 microM M1 metabolite, but not by 10 microM or less. The half maximal inhibition concentration of M1 metabolite was 24.8+/-9.8 microM in TM cells and 183+/-30.6 microM in CM cells. CONCLUSIONS: These findings indicate that there are CRAC channels in both TM and CM cells, and the influx of [Ca2+]i through the CRAC channels is suppressed by M1 metabolite. The inhibitory effect of M1 metabolite on CRAC currents was more prominent in TM cells than in CM cells. Suppression of the Ca2+ influx through CRAC channels by M1 metabolite is thought to regulate muscle tone in TM and CM cells. Therefore, CRAC inhibition by M1 metabolite may contribute to reduction of intraocular pressure.

Aniline Compounds↗
PubMed2006

[Construction of mutant myocilin plasmid and its expression in human trabecular meshwork cells].

PURPOSE: To establish the mutant Myocilin gene plasmid in order to study the function of Myocilin. METHODS: The mutant site was induced by site-directed mutagenesis. This plasmid were transfected into human trabecular meshwork cells (HTM cells) by cationic liposomes and the expression of Myocilin was examined by RT-PCR and Western blot analysis. RESULTS: The mutant plasmid were correctly constructed. Myocilin was efficiently expressed in HTM cells transfected with this plasmid. CONCLUSION: The constructed eukaryote expression plasmid pcDNA-MYOC-P370L could express Myocilin in HTM cells in vitro.

Cells, Cultured↗