Bovine Trabecular Meshwork Research Articles

Na-K-Cl cotransport regulates intracellular volume and monolayer permeability of trabecular meshwork cells

The trabecular meshwork (TM) of the eye plays a critical role in modulating intraocular pressure (IOP) through regulation of aqueous humor outflow, although the underlying mechanisms remain unknown. Ethacrynic acid, an agent known to inhibit Na-K-Cl cotransport of a number of cell types, recently has been reported to increase aqueous outflow and lower IOP through an unknown effect on the TM. In vascular endothelial cells and a variety of other cell types, the Na-K-Cl cotransporter functions to regulate intracellular volume. The present study was conducted to evaluate TM cells for the presence of Na-K-Cl cotransport activity and to test the hypothesis that modulation of cotransport activity alters intracellular volume and, consequently, permeability of the TM. We demonstrate here that bovine and human TM cells exhibit robust Na-K-Cl cotransport activity that is inhibited by bumetanide and by ethacrynic acid. Our studies also show that TM cell Na-K-Cl cotransport is modulated by a variety of hormones and neurotransmitters. Inhibition of the cotransporter either by bumetanide, ethacrynic acid, or inhibitory hormones reduces TM intracellular volume, whereas stimulatory hormones increase cell volume. In addition, shrinkage of the cells by hypertonic media stimulates cotransport activity and initiates a subsequent regulatory volume increase. Permeability of TM cell monolayers, assessed as transmonolayer flux of [14C]sucrose, is increased by hypertonicity-induced cell shrinkage and by bumetanide. These findings suggest that Na-K-Cl cotransport of TM cells is of central importance to regulation of intracellular volume and TM permeability. Defects of Na-K-Cl cotransport may underlie the pathophysiology of glaucoma.

Electrophysiological Properties of Cultured Human Trabecular Meshwork Cells

Previous studies using cultured bovine trabecular meshwork cells demonstrated at least two different cell types which were distinguishable morphologically and electrophysiologically. The purpose of the present study was to evaluate the electrical membrane properties of cultured human trabecular meshwork cells. Seven different human trabecular meshwork cell lines were grown from four different donors. One trabecular meshwork cell line was transformed by microinjection with SV40 DNA. The electrochemical properties of these TM cells were determined by micropuncture with glass microelectrodes. The mean membrane voltage at resting conditions differed between the cell lines (- 33·3 to - 58·7 mV). Application of 10-mmol l -1 Ba 2+ induced repetitive voltage spikes in all cell lines. The voltage transients similar to action potentials were inhibited by nifedipine, but insensitive to tetrodotoxin. Acetylcholine evoked depolarizations in three cell lines which were blocked by atropine. In one cell line isoproterenol caused sustained depolarizations sensitive to metipranolol. All three of the cell lines tested depolarized upon application of the vasoactive peptide endothelin-1. All untransformed cell lines showed voltage spikes typical for smooth muscle cells and functional receptors for endothelin-1 and cholinergic agonists. One out of three cell lines tested possessed β-adrenergic receptors influencing the membrane voltage.

Pilocarpine-induced lysosomal enzyme secretion, from bovine trabecular meshwork cells

It has long been held that pilocarpine-induced ciliary muscle contraction causes a mechanical change in the configuration of the trabecular meshwork, thereby increasing its fluid conductance. Though the results of many studies are consistent with this theory, other findings suggest a direct action of pilocarpine on the meshwork. We used cultured bovine trabecular meshwork (BTM) cells to study the cell biological effects of pilocarpine, chemically a weak base, and demonstrated increased vacuolization of the BTM cells after incubation with pilocarpine, reflecting a trapping of the protonated base inside the lysosomes. We also showed that pilocarpine enhanced the release of lysosomal hydrolases into the medium. We hypothesize that this is clinically relevant and that the pilocarpine-induced release of hydrolases modifies the extracellular matrix of the trabecular meshwork, thereby increasing its fluid conductance.

Studies of H2O2-Induced Effects on Cultured Bovine Trabecular Meshwork Cells

The trabecular meshwork is continuously challenged by oxidants that are both present in the aqueous humor and generated within the tissue. In this study we have investigated the antioxidant properties of cultured calf trabecular meshwork cells and evaluated the ability of the compound 4-hydroxy-2,2,6,6-tetramethypiperidine 1-oxyl (TEMPOL), a superoxide dismutase mimic, to prevent H2O2-induced cell damage. The cells were found to possess a high level of reduced glutathione, an undetectable amount of oxidized glutathione, and significant activities of glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and the hexose monophosphate shunt. The cells tolerated a 3-h exposure to a maintained, physiological level of H2O2 (0.02 mM); however, if the activity of glutathione reductase was inhibited, the same level of peroxide caused damage as indicated by cell contraction and blebbing. At a level of 0.05 mM H2O2, added to the medium as a single pulse, the shunt was stimulated eightfold and there were no significant effects on growth or morphology. However, a level of 0.1 mM H2O2 overwhelmed the antioxidant capability of the cells and produced severe effects. Treatment of the cells with TEMPOL prevented H2O2-induced inhibition of growth, formation of single-strand breaks in DNA, activation of the DNA-repair enzyme poly-ADP-ribose polymerase, and decrease in NAD, but TEMPOL was not able to prevent other changes such as the loss of GSH, decrease in glyceraldehyde-3-phosphate dehydrogenase activity, and stimulation of the shunt. Thus, certain intracellular effects of H2O2 in trabecular cells were shown to be caused directly by H2O2 whereas others were mediated through metal-catalyzed free radical reactions. The results indicate the presence of significant antioxidant activity in trabecular meshwork cells with a major contribution provided by the glutathione redox cycle.

Effects of Antiglaucoma Medications on Bovine Trabecular Meshwork Cells In Vitro

Using an in vitro culture system, we investigated the effects of five antiglaucoma drugs on growth and morphologic characteristics of bovine trabecular meshwork cells. Epinephrine hydrochloride (55-550 μM) and pilocarpine hydrochloride (0·8-16 mM), when added to the cultures for 3 days, inhibited trabecular cell growth in a dose-dependent manner. The lowest concentration at which the inhibitory effect was observed was 109 μM and 0·8 mM, respectively, for epinephrine and pilocarpine. Dipivefrin hydrochloride (26-260 μM), timolol maleate (116-1160 μM), and levobunolol hydrochloride (150-1500 μM) were also added to the cells for 3 days. These drugs caused a reduction in cell density, respectively, at concentrations higher than 103, 460, and 616 μM. Cell elongation was seen in cultures treated with epinephrine and dipivefrin, whereas levobunolol and timolol induced the cells to adopt a rounded appearance. Cells that had been exposed to pilocarpine were enlarged with numerous vacuoles. By scanning electron microscopic techniques, epinephrine, timolol, and levobunolol were found to retard the phagocytosis of latex beads by trabecular meshwork cells. Immunostaining with the use of antibodies to vimentin and actin revealed disorganization and condensation of cytoskeletal fibers in trabecular meshwork cells after treatment with epinephrine and dipivefrin. Little change was seen with comparable concentrations of a preservative, benzalkonium chloride, and a vehicle, Liquifilm tears. These results showed that antiglaucoma drugs, depending on their concentrations, may profoundly influence the growth and activity of trabecular meshwork cells.

Differential smooth muscle-like contractile properties of trabecular meshwork and ciliary muscle

The contractile properties of bovine trabecular meshwork and ciliary muscle strips were investigated using an electromagnetic force-length transducer for isometric force measurements. Acetylcholine, pilocarpine and aceclidine administration resulted in dose-dependent contractions of trabecular meshwork and ciliary muscle. Absolute forces were approximately 10 times larger in ciliary muscle than in trabecular meshwork. Maximal force evoked by aceclidine (5 × 10 −5 m), when compared to the pilocarpine (5 × 10 −5 m) response, was significantly higher in trabecular meshwork than in ciliary muscle. The results were 172·5 ± 12·6% ( n = 7) and 138·9±4·0% ( n = 8, P < 0·05), respectively. Depolarization induced by raised external potassium (120 m m), when compared to the acetylcholine response (10 −3 m), resulted in a small contraction of 19·3±4·2% in trabecular meshwork ( n = 5), and of 59·0±13·7% in ciliary muscle ( n = 4, P < 0·01). Both responses were inhibited by atropine (10 −5 m). The differential potassium effect may be explained by the large number of cholinergic nerve endings in ciliary muscle as compared to trabecular meshwork tissue. Recently, a dissociation between the effects of aceclidine on outflow resistance and accommodation has been described. Our data are consistent with these observations and provide evidence for a direct role of trabecular meshwork contractility in aqueous outflow regulation.

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 α-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 α-smooth muscle actin.

Chemoattractants produced by ocular cells induce trabecular meshwork cell migration

Media conditioned by bovine corneal endothelium and scleral fibroblasts were found to induce the migration of bovine trabecular meshwork cells. The migrational activity had both chemokinetic (stimulated random motion) and chemotactic (directional migration) components. That produced by corneal endothelial cells had a markedly more potent effect. The relevance of these in vitro studies is discussed. Biochemically stimulated migration and loss of meshwork cells may be involved in the pathogenesis of glaucoma.

Electrical and morphological evidence for heterogeneous populations of cultured bovine trabecular meshwork cells

Although trabecular meshwork cells are presumed to play an important role in determining ocular aqueous outflow resistance, little is known about their membrane transport characteristics. As in vivo access by microelectrodes is difficult, we used cell culture techniques to facilitate membrane voltage recording from cultured bovine trabecular meshwork cells. Phase-contrast microscopy revealed the presence of epithelial-like and spindle-shaped cell types. The mean membrane voltage for epithelial cells was −49·7±0·8 mV ( s.e.m., n = 143) and for spindle cells was −70·9±1·9 mV ( s.e.m., n = 48). These cells possess an electrogenic Na +/K +-ATPase and a K + conductance. K transference numbers ( t k) for [K +] from 5 to 80 m m were 0·50 for epithelial cells and 0·71 for spindle cells. The epithelial cells lack the electrogenic Na +/HCO 3 −-symport, thereby enabling their differentiation from corneal endothelial cells and confirming previous reports of differences between these cell types. A proportion of spindle cells demonstrated spontaneous and induced fluctuations of membrane voltage. One millimolar Ba 2+ ( n = 9) induced an immediate depolarization of membrane voltage, with the onset of “overshooting” action potentials, which were dependent on extracellualr Ca 2+ and Na + but were not blocked by tetrodotoxin, 10 −6 m. Spindle cells showed parallel alignment of intracellular smooth muscle specific α-isoactin filaments, whereas epithelial cells showed specks of non-fibrous staining. Electron microscopy revealed that epithelial cells had the characteristics of metabolically active cells, with few intermediate filaments (10–12 nm) and microfilaments (6–7 nm) and short cytoplasmic processes. Spindle cells had long cytoplasmic processes and abundant intermediate- and microfilaments. These data provide further evidence for multiple bovine trabecular cell types. The smooth muscle-like spindle cell may represent the previously proposed contractile element of the angle and its action could conceivably alter ocular outflow resistance.

Fibronectin induces migration of bovine trabecular meshwork cells in vitro

The migration of cultured bovine trabecular meshwork cells in response to fibronectin was studied using a 48-well migration chamber. Fibronectin was shown to be a potent chemoattractant for these cells, with a dose optimum of 30 μg ml −1. At this dose, the number of migrated cells was approximately 12 times that of the control, which was serum-free medium. Analysis of the migratory response revealed it to be mainly a chemokinesis, but chemotaxis was a substantial component. Electron microscopy demonstrated that adhesion and spreading of cells on the surface of the pore-containing membrane are necessary prerequisites to their migration through a pore. Microspikes are abundant during the movement of cellular processes into and through the pores. The migrated cells are extremely thin with a large surface area, but despite this phenotypic alteration the ultrastructure of migrated cells is unchanged.

Ascorbic acid modulates the production of fibronectin and laminin by cells from an eye tissue-trabecular meshwork

Ascorbic acid is a significant component in the aqueous humor of the eye, in which trabecular meshwork cells are immersed. Using immunostaining and Western blot analysis, the effects of ascorbic acid on the extracellular matrix production by cultured bovine trabecular meshwork cells were examined. It was found that, when the cultures were treated with 100, 250, and 500 μg/ml of ascorbic acid, the amount of fibronectin and laminin produced was enhanced. An enzyme-linked immunosorbent assay for quantitation further showed that the fibronectin and laminin levels in ascorbate-treated cultures were higher than those found in control cultures. These results indicate that ascorbic acid promotes fibronectin and laminin production and suggest that this substance may be one of the factors modulating basal lamina assembly in the trabecular meshwork.

Analysis of the proteins of calf and cow trabecular meshwork: Development of a model system to study aging effects and glaucoma

The proteins from the trabecular meshwork (TM) of calf and cow eyes were analysed to determine if differences in composition were present and to examine whether this tissue could be used as a framework for the study of glaucoma. Differences in polypeptide composition or amount of protein could be detected with extractions using either acetic acid, neutral buffers and urea, or guanidine hydrochloride. In general, the results suggest that an aggregation of proteins may be occurring with aging. The acid-soluble fraction of both calf and cow TM resembled older human TM specimens with the most prominent protein around 68 kD. To test the utility of the bovine TM system, a mixed function oxidation system was used to determine how the proteins of the TM would react to oxidative stress. Aggregation of the proteins in calf TM as well as actin could be demonstrated, consistent with the idea that the aggregation seen in the cow TM might be the result of oxidation of this tissue. The present study lays a foundation for future work on bovine TM and is consistent with the hypothesis that aging changes in this tissue might be a result of oxidative processes.

Effects of ascorbic acid on trabecular meshwork cells in culture

We examined the effects of ascorbic acid on trabecular meshwork cells in culture. Bovine trabecular meshwork cells were plated for 6 days and were exposed to media containing either no ascorbic acid or ascorbic acid in the following concentrations for 24 hr: 50-, 100-, 500-, and 750 micrograms ml-1. Fresh media were then added and incubation was continued for an additional 24 hr. The cell number was subsequently determined for the effects of the various concentrations of ascorbic acid on cell growth. In a parallel experiment, cells were labeled with [35S] sulfate (50 microCi ml-1) and [3H]glucosamine (15 microCi ml-1) for 24 hr with or without ascorbic acid for glycosaminoglycan studies. Results indicated that ascorbic acid stimulated the trabecular cell growth in culture at concentrations of 50-, 100-, and 250 micrograms ml-1. However, at 750 micrograms ml-1, cell growth was inhibited. In addition, cells exposed to 100 micrograms ml-1 of ascorbic acid demonstrated increased incorporation of [3H]glucosamine and [35S]sulfate; enhancement of incorporation of [35S]sulfate was also noted at 250 micrograms ml-1. Incorporation of labels was otherwise either unchanged or retarded at other concentrations. These data suggest that the ascorbic acid level in the aqueous humour may influence trabecular meshwork cell metabolism.

Cultured trabecular-meshwork cells: Immunohistochemical and lectin-binding characteristics

We examined the extracellular matrix formation and lectin-binding properties of cultured trabecular-meshwork cells established from cynomolgus monkey and bovine eyes. Using an avidin-biotin complex method, we found that the extracellular matrix in both the monkey and bovine cultures stained intensely with antibodies to fibronectin, type IV collagen, and laminin. These materials were especially prominent when the cultured monkey trabecular-meshwork cells aggregated in clusters, manifesting their close anatomic relationship to trabecular beams. Cell-surface and intracellular lectin-binding properties were studied using eight biotinylated lectins: concanavalin A (Con A), peanut agglutinin (PNA), Ricinus communis agglutinin (RCA), Dolichos biflorus agglutinin (DBA), soy bean agglutinin (SBA), Phaseolus vulgaris (PHA), wheat germ agglutinin (WGA), and Ulex europaeus agglutinin I (UEA I). Both monkey and bovine trabecular-meshwork cells showed positive cell surface and intracellular binding to Con A, WGA, and PHA. Only moderate cell-surface staining was observed with RCA, and no visible staining occurred with PNA, DBA, SBA, and UEA-1.

Lysosomal enzyme activities in cultured trabecularmeshwork cells

We examined the lysosomal enzyme levels in cultured trabecular meshwork cells. Histochemical studies revealed that bovine trabecular meshwork cells stained prominently for acid lipase, acid phosphatase, and acid esterase activities. Following phagocytic challenges, such as incubations with latex microspheres or zymosan particles, lysosomal enzyme staining was visibly enhanced. The uptake of foreign particles by trabecular-meshwork cells was evident. Data from biochemical assays for acid lipase, cholesteryl esterase, and acid esterase confirmed the histochemical findings. These results indicate that trabecular meshwork cells are actively phagocytic in culture and that lysosomal enzyme levels in these cells may be modulated by phagocytic challenge.

Timolol inhibits adenylate cyclase activity in the iris‐ciliary body and trabecular meshwork of the eye and blocks activation of the enzyme by salbutamol

The enzymatic activity of adenylate cyclase in homogenates and membrane-rich fractions prepared from rabbit iris-ciliary bodies and bovine trabecular meshwork was found to be inhibited by timolol. Treatment of iris-ciliary body homogenates with Triton X-305 resulted in abolition of the inhibitory effect of the drug on the activity of the enzyme. The stimulatory effect of salbutamol on the enzyme was also susceptible to blockade by timolol. It is suggested that the hypotensive action of timolol on intraocular pressure results from structural and functional changes induced on the plasma membranes of the iris-ciliary body and trabecular meshwork by the thiadiazole group of the molecule, and, also, from the occupation of the adrenergic receptors of the iris-ciliary body by the tert-butylamino-2-hydroxypropoxy part of the compound.

Some effects of phagocytosis on bovine meshwork cells in culture

In this tissue-culture study, some effects of phagocytosis on the cell were investigated. Third-passage bovine trabecular-meshwork cells were incubated with latex microspheres for periods of up to 60 hr. Cells which had been involved in phagocytosis were found to settle at a faster rate than control cells, but if microspheres were added to the medium of recently settled cells, there was a reduction in their proliferative activity. Incorporation of tritiated proline remained unchanged, whereas uptake of tritiated glucosamine increased with the time period of phagocytosis. The fibronectin scaffold was more extensive and elaborate when cells were pre-incubated with microspheres before settlement. Exposure to a low concentration of microspheres promoted a higher uptake of a second presentation of 125I-labelled microspheres — an effect which was not seen if the original dose was too high.

Phagocytosis of latex microspheres by bovine meshwork cells in culture.

The ability of cultured bovine trabecular meshwork cells to phagocytose latex microspheres was studied by electron microscopy. In addition, the incorporation of 125I gamma-globulin-coated microspheres by the meshwork cells was monitored. The cultured cells, on the basis of quantitation by scanning electron microscopy, did not show significant preference between biotic particles (gamma-globulin-coated microspheres) and abiotic particles (uncoated microspheres). Surface microvilli appeared to be involved in the initial stages of phagocytosis; indeed a significant negative correlation was demonstrated between the incidence of microvilli and the commitment to phagocytosis of preconfluent cells. Two weeks postconfluent cells were shown to be significantly less phagocytic than preconfluent cells. The importance of these findings in terms of the behaviour of meshwork cells in vivo is discussed.