Purpose: Recent evidence shows that much of the protein in the anterior chamber aqueous humor ent... more Purpose: Recent evidence shows that much of the protein in the anterior chamber aqueous humor enters diffusively through the root of the iris. The proximity of the protein entry point to the trabecular meshwork suggests that the protein content of the aqueous humor in the trabecular meshwork might be much higher than previously suggested. The authors were interested in investigating the possible hydrodynamic implications of these proteins. Methods: Bovine eyes were perfused with concentrations of bovine serum in buffer ranging from 0% to 15% to determine the effect on outflow resistance. Immunohistochemical methods were used on these eyes and unperfused eyes to determine the distribution of albumin in the anterior segment. Results: Preliminary perfusion studies suggested that increasing the concentration of serum in buffer from 0% to 15% decreased the rate of "wash-out" in bovine eyes, with a 15% solution essentially eliminating the wash-out phenomenon. Perfusion of a series of bovine eyes with a total of 5 ml of 15% serum in buffer showed a "wash-out" rate of 0.0498 +/- 0.0428 ([microliters/min/mm Hg]/[ml perfusate]), whereas control eyes perfused with buffer washed-out at a rate of 0.1677 +/- 0.0271 (P < 0.05); a second series of eyes perfused with a total of 10 ml of 15% serum washed-out at a rate of 0.0533 +/- 0.0294, whereas control eyes had a rate of 0.1813 +/- 0.0342 (P < 0.02). Immunohistochemical investigations showed significant quantities of albumin in the outflow pathway of unperfused eyes, whereas perfusion with buffer eliminated this protein; perfusion with 10% to 15% serum in buffer maintained the level of albumin in the outflow pathway similar to that found in unperfused eyes. Use of cuprolinic blue in a critical electrolyte concentration confirmed previous findings that sulfated proteoglycans are not eliminated from the trabecular meshwork during wash-out. Conclusions: Wash-out in nonhuman species may result from progressive depletion of an anterior segment depot of plasma-derived proteins entering the trabecular meshwork. Modeling studies confirm that plasma-derived proteins in the aqueous humor of the trabecular meshwork can generate a significant fraction of aqueous outflow resistance. The lack of wash-out in human eyes suggests that this system may maintain flow resistance in a fashion fundamentally different from other species.
Our goal was to evaluate the role of tyrosine phosphorylation in the complete formation of hemide... more Our goal was to evaluate the role of tyrosine phosphorylation in the complete formation of hemidesmosomes that occurs during development or during remodeling after injury. A corneal organ culture system was used to study hemidesmosome formation as it would occur in an intact tissue. Phosphorylation of the integrin subunit beta 4 and bullous pemphigoid antigen-1 (BPAG-1) was examined, as these proteins are known to play a role in linking the electron-dense plaques along the basal surface with the intermediate filaments to complete the formation of hemidesmosomes. Corneal epithelial sheets were placed on substrata that contained an intact basal lamina or basal laminae that had been either modified or removed. These constructs were incubated for up to 18 h, and hemidesmosome formation was evaluated by using transmission electron microscopy. When epithelial sheets were placed on intact basal laminae and incubated in the presence of the tyrosine kinase inhibitor genistein (200 microM), hemidesmosome formation was impaired. The formation of electron-dense regions was delayed, and no association of intermediate filaments was detected. Results were confirmed by biochemical studies. When the epithelium and underlying proteins were extracted and immunoprecipitated with beta 4 or BPAG-1, tyrosine phosphorylation decreased in the presence of genistein. In addition, the phosphorylation of beta 4 decreased when epithelial sheets were incubated on substrata from which the basal lamina had been removed or altered. Thus, a reduction in phosphorylation of tyrosine residues impairs the formation of mature hemidesmosomes, and substrata that fail to support hemidesmosome formation also demonstrate decreased phosphorylation of tyrosine residues.
ABSTRACT Previous studies have suggested that hydrophobic moieties within the aqueous outflow cha... more ABSTRACT Previous studies have suggested that hydrophobic moieties within the aqueous outflow channels might interact with certain aqueous components to retard outflow. While elastin is among the most hydrophobic proteins in the trabecular meshwork, it reacts poorly with conventional ultrastructural staining methods, so its potential role in regulating outflow could not be assessed. It was our goal to specifically localize elastin ultrastructurally using polyclonal antibodies against alpha elastin and its soluble precursor, tropoelastin. Human aorta served as a positive control. Preadsorption of the primary antibodies or their substitution with either normal rabbit serum or Tris buffer resulted in negligible labelling. With either antibody, only the electron-lucent elements in the center of elastic fibers of the trabecular meshwork were labelled, indicating that only these elements truly represent elastin. The pattern of elastin distribution within these fibers is most consistent with that found in tendons elsewhere in the body.
Purpose: Recent evidence shows that much of the protein in the anterior chamber aqueous humor ent... more Purpose: Recent evidence shows that much of the protein in the anterior chamber aqueous humor enters diffusively through the root of the iris. The proximity of the protein entry point to the trabecular meshwork suggests that the protein content of the aqueous humor in the trabecular meshwork might be much higher than previously suggested. The authors were interested in investigating the possible hydrodynamic implications of these proteins. Methods: Bovine eyes were perfused with concentrations of bovine serum in buffer ranging from 0% to 15% to determine the effect on outflow resistance. Immunohistochemical methods were used on these eyes and unperfused eyes to determine the distribution of albumin in the anterior segment. Results: Preliminary perfusion studies suggested that increasing the concentration of serum in buffer from 0% to 15% decreased the rate of "wash-out" in bovine eyes, with a 15% solution essentially eliminating the wash-out phenomenon. Perfusion of a series of bovine eyes with a total of 5 ml of 15% serum in buffer showed a "wash-out" rate of 0.0498 +/- 0.0428 ([microliters/min/mm Hg]/[ml perfusate]), whereas control eyes perfused with buffer washed-out at a rate of 0.1677 +/- 0.0271 (P < 0.05); a second series of eyes perfused with a total of 10 ml of 15% serum washed-out at a rate of 0.0533 +/- 0.0294, whereas control eyes had a rate of 0.1813 +/- 0.0342 (P < 0.02). Immunohistochemical investigations showed significant quantities of albumin in the outflow pathway of unperfused eyes, whereas perfusion with buffer eliminated this protein; perfusion with 10% to 15% serum in buffer maintained the level of albumin in the outflow pathway similar to that found in unperfused eyes. Use of cuprolinic blue in a critical electrolyte concentration confirmed previous findings that sulfated proteoglycans are not eliminated from the trabecular meshwork during wash-out. Conclusions: Wash-out in nonhuman species may result from progressive depletion of an anterior segment depot of plasma-derived proteins entering the trabecular meshwork. Modeling studies confirm that plasma-derived proteins in the aqueous humor of the trabecular meshwork can generate a significant fraction of aqueous outflow resistance. The lack of wash-out in human eyes suggests that this system may maintain flow resistance in a fashion fundamentally different from other species.
Our goal was to evaluate the role of tyrosine phosphorylation in the complete formation of hemide... more Our goal was to evaluate the role of tyrosine phosphorylation in the complete formation of hemidesmosomes that occurs during development or during remodeling after injury. A corneal organ culture system was used to study hemidesmosome formation as it would occur in an intact tissue. Phosphorylation of the integrin subunit beta 4 and bullous pemphigoid antigen-1 (BPAG-1) was examined, as these proteins are known to play a role in linking the electron-dense plaques along the basal surface with the intermediate filaments to complete the formation of hemidesmosomes. Corneal epithelial sheets were placed on substrata that contained an intact basal lamina or basal laminae that had been either modified or removed. These constructs were incubated for up to 18 h, and hemidesmosome formation was evaluated by using transmission electron microscopy. When epithelial sheets were placed on intact basal laminae and incubated in the presence of the tyrosine kinase inhibitor genistein (200 microM), hemidesmosome formation was impaired. The formation of electron-dense regions was delayed, and no association of intermediate filaments was detected. Results were confirmed by biochemical studies. When the epithelium and underlying proteins were extracted and immunoprecipitated with beta 4 or BPAG-1, tyrosine phosphorylation decreased in the presence of genistein. In addition, the phosphorylation of beta 4 decreased when epithelial sheets were incubated on substrata from which the basal lamina had been removed or altered. Thus, a reduction in phosphorylation of tyrosine residues impairs the formation of mature hemidesmosomes, and substrata that fail to support hemidesmosome formation also demonstrate decreased phosphorylation of tyrosine residues.
ABSTRACT Previous studies have suggested that hydrophobic moieties within the aqueous outflow cha... more ABSTRACT Previous studies have suggested that hydrophobic moieties within the aqueous outflow channels might interact with certain aqueous components to retard outflow. While elastin is among the most hydrophobic proteins in the trabecular meshwork, it reacts poorly with conventional ultrastructural staining methods, so its potential role in regulating outflow could not be assessed. It was our goal to specifically localize elastin ultrastructurally using polyclonal antibodies against alpha elastin and its soluble precursor, tropoelastin. Human aorta served as a positive control. Preadsorption of the primary antibodies or their substitution with either normal rabbit serum or Tris buffer resulted in negligible labelling. With either antibody, only the electron-lucent elements in the center of elastic fibers of the trabecular meshwork were labelled, indicating that only these elements truly represent elastin. The pattern of elastin distribution within these fibers is most consistent with that found in tendons elsewhere in the body.
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