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D L Epstein

Publications and source records attributed to D L Epstein.

At least 55 records · Page 3Linked to original sources
PubMed1996

Ethacrynic acid disrupts steady state microtubules in vitro.

PURPOSE: Ethacrynic acid (ECA) has been shown to increase facility of aqueous outflow in whole eyes and perfused anterior segments, to open up spaces between cells in the trabecular meshwork and inner wall of Schlemm's canal, and to cause separation and retraction of trabecular meshwork and endothelial cells in culture. One mechanism by which ECA has been proposed to act in cells is via disruption of microtubules, leading to cell retraction. Although it is known that ECA can inhibit de novo assembly of microtubules from tubulin subunits in vitro, we wanted to determine, as a better correlate to the proposed effect of ECA in cells, whether ECA could disrupt microtubule polymers that had reached steady state. We also wanted to determine whether calcium ion could enhance this process. METHODS: We therefore assembled purified and crude porcine brain tubulin to steady state at 37 degrees C and then added ECA and/or calcium. Reaction kinetics were followed spectrophotometrically. RESULTS: We found that ECA effectively disrupted assembled microtubules in vitro. Although 0.8-1.0 mM ECA was required to produce a half-maximal effect in pure tubulin microtubules and 0.2-0.3 mM ECA was necessary with crude microtubule protein, significant disassembly also occurred in the 0.01-0.2 mM range. Calcium had a greater maximal effect than ECA, and was more potent on a molar basis, showing half maximal effect between 2 and 12 microM free calcium ion. Combination experiments showed that ECA did not act synergistically with calcium to increase microtubule disassembly. CONCLUSIONS: Our results are consistent with the proposed disruptive action of ECA on the assembled microtubules of outflow pathway cells, but do not support a rise in intracellular calcium as being an added factor.

Animals↗
PubMed1996

Effect of age on superoxide dismutase activity of human trabecular meshwork.

PURPOSE: It has been hypothesized that accelerated aging of the trabecular meshwork, perhaps because of oxidative damage, is involved in the pathogenesis of primary open angle glaucoma. The authors sought to evaluate the effect of donor age on the specific activity of superoxide dismutase and catalase in normal fresh human cadaver trabecular meshwork. METHODS: Total superoxide dismutase and catalase were assayed in tissue extracts generated from fresh human trabecular meshwork. Cadaver tissue was obtained from 19 donors (18 paired) of a wide age range (30 to 91 years). The assays were performed within 6 hours of enucleation and within 36 hours of donor death. Enzyme-specific activities were calculated using protein concentration of the extract as the denominator. RESULTS: Multiple regression analysis modeled with age and time from death until the beginning of the experiment was performed. The specific activity of superoxide dismutase declined with age (P = 0.00022; r2 = 0.67). There was no effect of age on catalase specific activity (P = 0.24; r2 = 0.16). The time from donor death until the beginning of the experiment was not a significant factor (P > 0.28). CONCLUSIONS: The specific activity of superoxide dismutase, but not catalase, demonstrates an age-dependent decline in normal cadaver human trabecular meshwork. The potential role of superoxide dismutase in primary open angle glaucoma, a disorder of the aging trabecular meshwork, warrants further investigation.

Adult↗
PubMed1996

Comparative study of the efficacy of argon laser trabeculoplasty for exfoliation and primary open-angle glaucoma.

PURPOSE: To investigate the efficacy of argon laser trabeculoplasty (ALT) for the treatment of primary open-angle glaucoma (POAG) and of glaucoma associated with exfoliation syndrome (EXF). METHODS: Review of > 200 charts from patients treated with ALT between 1981 and 1987 identified 66 POAG and 29 EXF eyes that underwent initial 180 degrees treatment. Variables including baseline intraocular pressure (IOP), age, sex, angle pigmentation, and follow-up IOP were studied with numerous statistical analyses. Multiple failure modes were used to define failure rate. All POAG and EXF patients were white. RESULTS: The baseline pre-ALT IOP was 23.2 +/- 6.1 mm Hg for the POAG group and 25.8 +/- 5.9 mm Hg for the EXF group (p < .06). Mean follow-up time was 27 +/- 22 months for POAG eyes and 23 +/- 20 months for EXF eyes. Using failure mode 4 (glaucoma surgery, third laser, IOP < or = 22 mm Hg, or two consecutive IOPs > 85% of original baseline IOP), the 1-year failure rates were 40% (POAG) and 18% (EXF), and the 3-year rates were 58% (POAG) and 47% (EXF), p < 0.89 by log-rank test. A Cox proportional hazards model controlling for baseline IOP, age, sex, and angle pigmentation demonstrated that exfoliation status did not affect progression to filtering surgery (p > 0.60). CONCLUSION: Using multiple failure modes, the results suggest that the success rate of ALT in exfoliation glaucoma tends to decrease over time, but then stabilizes at this reduced value at a level similar to that of POAG. By 3 years, there is a substantial failure rate in both POAG eyes and EXF eyes. Although the initial response to ALT in EXF patients is greater, the long-term outcome is similar for both groups.

Exfoliation Syndrome↗
PubMed1994

Interaction of ethacrynic acid with bovine brain tubulin.

Ethacrynic acid is a diuretic agent that reacts with sulfhydryl groups in proteins, and which shows promise of effectiveness in the treatment of glaucoma. Ethacrynic acid is a known inhibitor of microtubule assembly in vitro (Xu et al., Arch Biochem Biophys 296: 462-67, 1992). We have used N,N'-ethylenebis (iodoacetamide) (EBI) as a probe to examine the sulfhydryl groups of tubulin; EBI can form two intra-chain cross-links in beta-tubulin. One of these, beta*, connects Cys239 with Cys354; the other, beta s, joins Cys12 with either Cys201 or Cys211 (Little and Ludueña, EMBO J 4: 51-56, 1985; Biochim Biophys Acta 912: 28-33, 1987). Formation of beta * inhibits microtubule assembly in vitro, consistent with the hypothesis that Cys239 has an assembly-critical sulfhydryl (Bai et al., Biochemistry 28: 5606-5612, 1989). We have examined the interaction of ethacrynic acid with the sulfhydryl groups of bovine brain tubulin. We found that 130 microM ethacrynic acid gave half-maximal inhibition of assembly, but had no effect on the formation of the beta * cross-link by EBI. Ethacrynic acid, however, did inhibit substantially formation of the beta s cross-link at this concentration and half-maximally inhibited it at approximately 185 microM. Half-maximal inhibition of the alkylation of tubulin sulfhydryls by iodo [14C]acetamide was obtained at an ethacrynic acid concentration in the range of 190-325 microM. These results indicate that ethacrynic acid can inhibit microtubule assembly by reacting with sulfhydryl groups other than those of Cys239 and Cys354 and suggest that other sulfhydryl groups in tubulin could be assembly-critical. These results also raise the possibility that these other assembly-critical sulfhydryls may be those of Cys12, Cys201 or Cys211.

Alkylation↗
PubMed1994

Increased prevalence of occludable angles and angle-closure glaucoma in patients with pseudoexfoliation.

The association between angle-closure glaucoma and pseudoexfoliation is controversial. We retrospectively studied the angle configurations of 54 patients with pseudoexfoliation and found gonioscopically occludable angles in five cases (9.3%). We also analyzed the data from several large studies of pseudoexfoliation patients and found an increased prevalence of acute angle-closure glaucoma in this group. These data suggest that pseudoexfoliation patients may represent a high-risk population for the development of angle-closure glaucoma.

Acute Disease↗
PubMed1993

Is primary open angle glaucoma caused by small proteins?

Primary open angle glaucoma is a disease which is characterized by a decreased facility of outflow of aqueous humor from the anterior segment of the eye. Analyses by gel exclusion chromatography of the aqueous humor of normal monkeys have shown that the proteins in this fluid range in size from approximately 40-200 kilodaltons. Although small proteins and peptides are seen in the aqueous humor by SDS gel electrophoresis, these smaller proteins appear to elute by column chromatography with the albumin peak. In the outflow of the aqueous humor, the trabecular meshwork may be an efficient sieve for proteins of intermediate size, but this tissue may be obstructed if the proteins present become too large or too small as a result of other disease processes in the eye. Primary open angle glaucoma may be the result of the inability of the trabecular meshwork to filter small proteins and polypeptides efficiently.

Animals↗
PubMed1992

Ethacrynic acid inhibition of microtubule assembly in vitro.

Ethacrynic acid (ECA) is a sulfhydryl reactive diuretic drug. Recent studies show that ocular administration of ECA may have potential efficacy for treatment of glaucoma. ECA affects cell shape in cultured cells from the eye outflow pathway and the microtubule system is disrupted. We have studied the effect of ECA on microtubule protein (MTP) (tubulin and microtubule-associated proteins) and purified tubulin assembly. Fifty percent inhibition of MTP (1.8 mg/ml) assembly was found at 70 microM ECA in buffer and 410 microM ECA in 30% glycerol in buffer. If all sulfhydryl groups were attributed to tubulin, then approximately two sulfhydryls were blocked at 50% inhibition. Tubulin (2 mg/ml) assembly showed 50% inhibition at 175 microM ECA and approximately 2 sulfhydryl groups were lost. Increasing ECA preincubation times (0-60 min) with tubulin showed that the longer the preincubation time, the longer the lag time, and the slower the rate of assembly and that the percentage of inhibition was proportional to the ECA preincubation time. The number of blocked sulfhydryls also increased with preincubation time. Approximately two sulfhydryls were blocked at 50% inhibition of assembly. The critical concentration for assembly increased twofold when tubulin was preincubated with 0.1 mM ECA, suggesting a loss of active tubulin. Fifty percent inhibition of taxol-induced MTP and tubulin assembly occurred at 190 and 280 microM ECA, respectively, with 3.6 to 3.8 sulfhydryls blocked, respectively. Taxol protects microtubules from disassembly by ECA, suggesting that the ECA binding key sulfhydryls are blocked in the microtubule. These results suggest that ECA reacts slowly with tubulin and blocks sulfhydryl groups important for assembly. Microtubule-associated proteins and glycerol protect the sulfhydryls and so more ECA is necessary to inhibit assembly. Since the number of blocked sulfhydryls is greater at 50% inhibition for taxol-induced microtubules, sulfhydryl blocked tubulin incompetent to assemble under normal conditions may be induced to do so with taxol.

Alkaloids↗
PubMed1992

The effect of intracameral ethacrynic acid on the intraocular pressure of living monkeys.

Previous studies have shown that the sulfhydryl-reactive ethacrynic acid increases outflow facility in living monkeys when perfused via the anterior chamber. To study its potential clinical use further, living monkeys were intracamerally injected with 10 microliters of ethacrynic acid, with concentrations ranging from 0.5 to 7.5 mmol/l. The fellow control eye was injected with 10 microliters of diluent. The status of the anterior segment was monitored by slit-lamp biomicroscopy and the intraocular pressure was measured by pneumatonometry with the monkeys anesthetized with ketamine. The anterior segment of living monkeys tolerated injections up to 3.0-mmol/l ethacrynic acid without marked adverse effects. One of 13 monkey eyes injected with 3.0-mmol/l ethacrynic acid demonstrated mild reversible segmental corneal edema. The greatest mean intraocular pressure reduction in the 3.0- to 3.75-mmol/l group occurred at six hours, with the experimental intraocular pressure decreasing 2.9 mm Hg compared to a mean intraocular pressure increase of 0.1 mm Hg in the control group (n = 19). Concentrations of ethacrynic acid less than 3.0 mmol/l did not provide reliable reduction of intraocular pressure, whereas concentrations greater than 3.75 mmol/l caused a greater incidence and severity of corneal edema. We believe that the intracameral injection of ethacrynic acid can reliably and safely reduce intraocular pressure in living monkey eyes, and that this drug deserves further investigation as a potential antiglaucomatous agent.

Animals↗
PubMed1992

The effect of intracamerally injected ethacrynic acid on intraocular pressure in patients with glaucoma.

We studied the effect of ethacrynic acid on intraocular pressure in eyes with advanced open-angle glaucoma. Five to 15 microliters of ethacrynic acid (3.3 to 9.8 micrograms) was injected intracamerally after retrobulbar anesthesia was achieved in five eyes of five patients with advanced glaucoma. Intraocular pressure before treatment ranged from 26 to 46 mm Hg with maximal medical treatment. A reduction in intraocular pressure from 9 to 31 mm Hg was observed in all patients three to 24 hours after treatment, and this effect lasted for three days, with a gradual return of intraocular pressure to pretreatment values one week after treatment. No acute corneal or anterior chamber side effects were observed and results of corneal endothelial cells counts were essentially unchanged two months after treatment. We suggest that ethacrynic acid may represent a new class of antiglaucoma medication. Intracameral administration of ethacrynic acid or a derivative might be contemplated in the future at the time of other ocular surgical procedures to treat short-term intraocular pressure increase.

Adult↗
PubMed1992

Ethacrynic acid increases facility of outflow in the human eye in vitro.

Anterior segments of human donor eyes were perfused with culture medium at a perfusion pressure of 15 mm Hg in a 5% carbon dioxide environment at 37 degrees C. After determination of a baseline facility of outflow, the perfusion chamber contents were exchanged with either drug vehicle or ethacrynic acid, at concentrations ranging from 0.01 to 0.25 mmol/L, after which postdrug facility was measured in the continuous presence of drug vehicle or ethacrynic acid. Ethacrynic acid increased facility of outflow from 28% to 105% at ethacrynic acid concentrations of 0.01 to 0.25 mmol/L, respectively. No morphologic correlate of the facility increase was observed with 0.01-mmol/L ethacrynic acid, nor were there any signs of cellular toxic effects. At 0.1 mmol/L, separations between trabecular meshwork cells and breaks between inner-wall cells were observed. At 0.25 mmol/L, focal areas of cell swelling and necrosis were noted. This study demonstrated that ethacrynic acid increases outflow facility in the aged human eye at concentrations that produce no apparent toxic effects. Therefore, ethacrynic acid may potentially prove useful in the treatment of glaucoma.

Adult↗
PubMed1992

Effects of topical ethacrynic acid adducts on intraocular pressure in rabbits and monkeys.

We evaluated the effect of topical ethacrynic acid on rabbit and monkey intraocular pressure. In a preliminary experiment, 100-mmol/L ethacrynic acid applied topically to Dutch-Belted rabbit eyes was associated with an 8-mm Hg lowering of intraocular pressure. However, corneal edema was severe, and the corneal epithelium sloughed off. To try to maintain the pressure-lowering effect but reduce the corneal side effects, we attempted to create an adduct of ethacrynic acid by utilizing ethacrynic acid's sulfhydryl reactivity. Ethacrynic acid was mixed with equimolar cysteine to bind the sulfhydryl-reactive sites on ethacrynic acid. The goal was to expose the cornea to adducted ethacrynic acid, which might then dissociate in the anterior chamber via a retro-Michael reaction. Intraocular pressure decreased 8.9 mm Hg (n = 40) with this treatment, and corneal edema was lessened (32 of 40 eyes had mild to no edema). However, we observed that when the eye was treated before ethacrynic acid-cysteine administration with topical acetylcysteine, the corneal side effects were reduced further and the intraocular pressure effect remained. In living cynomolgus monkeys receiving a single pretreatment drop of 75-mmol/L acetylcysteine followed by two drops of 130-mmol/L ethacrynic acid and 130-mmol/L cysteine, an intraocular pressure lowering of 9.9 mm Hg was observed (n = 7). However, in three of seven eyes corneal edema developed. Pretreatment with two drops of acetylcysteine eliminated the pressure-lowering effect but did not confer any added corneal protection. Our results indicate that topical ethacrynic acid-cysteine is effective in lowering the intraocular pressure of rabbits and cynomolgus monkeys and, when combined with acetylcysteine pretreatment, may offer the potential for a new topical therapeutic regimen for use in glaucoma.

Acetylcysteine↗
PubMed1992

Cardiopathic effects of dichloroacetate in the fetal Long-Evans rat.

Dichloroacetic acid (DCA) is a by-product of the chlorine disinfection of water and may occur in treated water at levels exceeding 100 micrograms/L. Previous studies revealed teratogenic effects, particularly heart malformations, at high doses (900-2,400 mg/kg given on days 6-15 of pregnancy). In a series of three studies, groups of 7-10 Long-Evans rats were dosed with 1,900 mg/kg of DCA on days 6-8, 9-11, or 12-15; with 2,400 mg/kg on days 10, 11, 12, or 13; and with 3,500 mg/kg on days 9, 10, 11, 12, or 13, in an attempt to determine the most sensitive period and further characterize the heart defect. In a fourth study, six dams were treated with 1,900 mg/kg of DCA days 6-15 of pregnancy, and 56 fetuses were harvested for light microscopy of the heart. Eight control fetuses from four litters were also examined. No heart malformations were seen in the groups treated with 1,900 mg/kg DCA days 6-8 but were present in the group treated on days 9-11 and 12-15, with the higher incidence occurring on days 12-15. Single doses of 2,400 mg/kg DCA given on days 10, 11, 12, or 13 resulted in a much lower incidence of cardiac malformations, which occurred only on days 10 and 12. The high dose of DCA (3,500 mg/kg) did not increase the incidence of heart defects but showed that dosing on day 9 as well as on days 10 and 12 would produce the defect. The defects seen were characterized as high interventricular septal defects (H-IVSD). Light microscopy showed that the defect was caudal to the semilunar valves, with the anterior right wall of the aorta communicating with the right ventricle. Another aspect of the defect is at the level of the semilunar valves, with the right cusp or sinus of Valsalva in communication with the right ventricle. The defects are discussed more fully and methods for further study suggested.

Animals↗
PubMed1992

Protein analysis of monkey aqueous humor.

Aqueous humor from individual eyes of young monkeys (Macaca mulatta) was analyzed by gel exclusion chromatography, and one- and two-dimensional gel electrophoresis. The data indicated that in young normal monkey the main peak of protein eluting from gel exclusion columns was observed around 80,000 daltons. There was a small amount of heavy molecular weight material eluting from these columns with an apparent molecular size of greater than 500,000 daltons, but very little material smaller than 40,000 daltons. By one- and two-dimensional gel electrophoresis, the major polypeptide components in the monkey aqueous were similar to those reported for the human aqueous with polypeptides at around 170, 130, 110, 80, 67, 60, 42, 34, 28, 25, 22, 16, and 14 kilodaltons (kD). The monkey aqueous humor also contains the protease inhibitor cystatin.

Animals↗
PubMed1992

Thiol adducts of ethacrynic acid increase outflow facility in enucleated calf eyes.

Ethacrynic acid (ECA), a sulfhydryl (SH)-reactive diuretic drug, has been shown to increase outflow facility (C) both in living monkey eyes and in the calf eye, in vitro (Epstein et al. 1987). In an attempt to increase the therapeutic index of this drug for potential clinical use in glaucoma, we explored the effect of various thiol adducts of ECA on C in the calf eye in vitro. These adducts might be expected to liberate ECA by a reversible retro-Michael type reaction. Enucleated calf eyes were perfused at 25 degrees C at 15 mm Hg for 5 hours with various ECA-thiol adducts. ECA-cysteine at 0.25 mM (for each) increased outflow facility 104% compared to 38% in sham manipulated eyes (n-10; p less than .005). A dose response effect was demonstrated from 0.01 mM to 0.25 mM. A relative potency table (for increasing C) was established for several ECA-thiol adducts: Cysteine = cysteamine greater than glutathione greater than N-Acetyl cysteine greater than thiosalicylic acid greater than N-Acetyl cysteamine. This study identifies the potential of utilizing various derivatives of ECA as outflow pathway acting agents.

Animals↗
PubMed1992

The relationship between pore density and outflow facility in human eyes.

The inner wall (IW) endothelial lining of Schlemm's canal was examined in six normal human eyes and four eyes with primary open angle glaucoma (POAG). Outflow facility was measured using constant pressure perfusion at 15 mmHg, eyes were fixed at 15 mmHg, and the IW endothelial lining was isolated and examined by scanning electron microscopy. Pore density, pore diameter, and bulge density were recorded by quadrant, and pore size and density were used to estimate IW endothelial facility, resistance, and hydraulic conductivity (facility per unit area). In POAG eyes, pores were less common (489 +/- 172 vs 1437 +/- 423 pores/mm2; P less than .005) and appeared to be more unevenly distributed than in normal eyes. A regional analysis of pore density (by quadrant) failed to detect a significant difference between quadrants of normal or POAG eyes. Pore density was correlated with measured outflow facility in normal eyes alone (P less than .02) and when normal eyes were pooled with POAG eyes (P less than .001). The percentage of total resistance attributed to the IW endothelium was 5.8% in normals compared to 9.5% in POAG eyes. This indicates there is a greater pressure drop across the IW endothelium in POAG eyes, suggesting that an intrinsic difference in IW endothelial function exists between normal and glaucomatous eyes. However, this difference alone does not account for the decreased outflow facility in POAG eyes. IW endothelial hydraulic conductivity is markedly higher than that of other vascular endothelia. We hypothesize that this may protect the IW endothelial lining of Schlemm's canal from mechanical stress induced by the relatively high rate of transcellular fluid flow.

Aged↗
PubMed1992

Ethacrynic acid induces reversible shape and cytoskeletal changes in cultured cells.

Cell cultures derived from trabecular meshworks of human and bovine eyes and from bovine vascular endothelia were incubated at 37 degrees C for 1 hr with ethacrynic acid (ECA, 0.1-0.5 mmol/l) dissolved in culture medium. At 2 hr after the initial exposure, ECA at concentrations up to 0.4 mmol/l induced a reversible alteration in cell shape in all three cell types that was coincident with a change in the staining pattern of major cytoskeletal components including actin, alpha-actinin, vinculin, and vimentin. Distinct progressive alterations in beta-tubulin also occurred, with initial changes observed 10 min after ECA exposure. The ECA-induced changes in tubulin were blocked in part by preincubation with taxol (which stabilizes the microtubule structure), but they appeared to differ from those occurring with nocodazole (which interferes with tubulin assembly). These results suggest the possibility that ECA-induced increases in outflow facility may be mediated by alterations in the cytoskeletons of outflow pathway cells.

Adolescent↗
PubMed1992

Localization of smooth muscle and nonmuscle actin isoforms in the human aqueous outflow pathway.

alpha-Smooth muscle actin is the isoform of actin restricted to vascular smooth muscle, pericytes, myofibroblasts and, certain other cells that are of myoid origin. We investigated the distribution of alpha-smooth muscle actin and nonmuscle specific filamentous actin in the human aqueous outflow system by immunohistochemical methods. Filamentous actin was observed in all cellular constituents of the outflow pathway, while distribution of alpha-smooth muscle actin was restricted to the ciliary muscle, to specific cells throughout the trabecular meshwork, and to cells adjacent to the outer wall and the collector channels. The ciliary muscle extended deep into the corneoscleral meshwork, far anterior to the scleral spur. These findings agree with our previous study localizing the distribution of smooth muscle myosin in the human aqueous outflow pathway. Although functionality of the immunoreactive cells needs to be demonstrated, our data show that a potentially contractile apparatus exists in a subpopulation of trabecular meshwork cells and in certain cells of the more distal components of the outflow system.

Actins↗