摘要:

PurposeEthacrynic 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 inhibitde novoassembly of microtubules from tubulin subunitsin 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 deter-mine whether calcium ion could enhance this process.MethodsWe therefore assembled purified and crude porcine brain tubulin to steady state at 37°C and then added ECA and/or calcium. Reaction kinetics were followed spectrophotometrically.ResultsWe 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 12µM free calcium ion. Combination experiments showed that ECA did not act synergistically with calcium to increase microtubule disassembly.ConclusionsOur 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. Curr. Eye Res. 15: 985–990, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017644

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeExtracellular matrix (ECM) plays a major role in the development and regeneration of various epithelial cells including retinal pigment epithelium (RPE), and attachment to ECM inhibits RPE apoptosis. Transplantation of ECM prior to the transplantation of RPE may modulate the survival and subsequent proliferation of transplanted RPE. Thus, we have developed a technique to harvest and transfer native ECM produced by bovine, porcine and human cell lines.MethodsECM was prepared by treating a confluent monolayer of cells with 0.02 N ammonium hydroxide. The ECM was then coated with a thin 100µlayer of 12% gelatin and cooled to 4±C. Patches of the ECM were isolated and transferred to another culture plate. The transferred ECM was characterized by immunohistochemistry. We determined the ability of cultured RPE to reattach to the harvested ECM, and the ability of the harvested ECM to inhibit RPE apoptosis.ResultsNative ECM can be transferred to another location en bloc with this technique. Immunohistochemistry demonstrates that the transferred ECM contains fibronectin, laminin and collagen IV. The reattachment rate of human RPE to each type of transferred ECM is higher (83.6±2.8%) than RPE reattachment to bare tissue culture plastic (57.6±9.8%). The apoptotic rate of attached RPE cells on transferred bovine corneal endothelial ECM (4.3±1.4%) is lower than their apoptotic rate on bare plastic (69.3±4.1%). The apoptotic rates of unattached cells are 80.3±4.4% on transferred bovine corneal endothelial ECM and 79.2±3.4% on bare plastic.ConclusionsWe conclude that ECM produced by various cell lines can be harvested and transferred by this technique. The transferred ECM promotes cell reattachment and inhibits RPE cell apoptosis. Harvesting and transfer of ECM at the time of RPE transplantation may inhibit apoptosis and promote survival of the transplant. Curr. Eye Res. 15: 991-997, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017645

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor

摘要:

PurposeThe primary aim of this study was to develop and characterize a simple flow cytometric method of quantifying rod outer segment (ROS) phagocytosis in cultured retinal pigment epithelial (RPE) cells. A secondary aim was to compare the kinetics of ROS phagocytosis in an immortal human RPE cell line with untransformed human RPE cells.MethodsFlow cytometry was performed on RPE cells that had been challenged with fluorescein isothiocyanate-labeled ROS (FITC-ROS) and phagocytosis was calculated by subtracting background cellular autofluorescence.ResultsNon-specific uptake of fluorescent label was negligible and RPE cells phagocytosed FITC-ROS and unlabeled ROS with equal efficacy. The kinetics of FITC-ROS phagocytosis in the D407 RPE cell line differed from early passage untransformed human RPE cultures. FITC-ROS phagocytosis proceeded at a fairly linear rate for the first 12 h in the 3 human cell cultures studied, but was rapid for the first 3 h before slowing in the D407 cells. Within all cell populations, there was a heterogeneity of phagocytic activity which varied with time.ConclusionsThis automated technique for measuring phagocytosis is rapid, simple, highly accurate, avoids radiation hazards, and permits study of heterogeneity within cell populations. The biochemistry, physiology and pathophysiology of the interactions between retinal pigment epithelial (RPE) cells and photoreceptors continue to be areas of considerable research interest (1, 2, 3). Vital to such work is the ability to accurately quantify rod outer segment (ROS) phagocytosis by RPE cells. Current in vitro techniques of measuring ROS phagocytosis use either automated or manual methods to count phagosomes. While manual counting techniques offer the advantage of visual quality control, they are highly labor intensive, there is a practical limitation to the number of phagosomes that can be counted, and measurements suffer from relatively large standard errors (3). Automated methods include scintillation counting and flow cytometry. Problems with radiolabels include radiation hazards, nonspecific radiolabel uptake, and limited visual control (3). Flow cytometry, on the other hand, circumvents nearly all of these problems and may prove to be the optimal phagocytosis assay. Curr. Eye Res. 15: 998-1003, 1996.

ISSN:0271-3683    

DOI:10.3109/02713689609017646

出版商:Taylor&Francis    

年代:1996

数据来源: Taylor