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Your search keyword '"Pales J"' showing total 2 results
Start Over Author "Pales J" Topic trabecular meshwork
2 results on '"Pales J"'

1. Use of transduction proteins to target trabecular meshwork cells: outflow modulation by profilin I.

Author

Gomez-Cabrero A, Comes N, Gonzalez-Linares J, de Lapuente J, Borras M, Pales J, Gual A, Gasull X, and Morales M

Subjects
Actins metabolism, Animals, Aqueous Humor metabolism, Blotting, Western, Cattle, Cells, Cultured, Cloning, Molecular, Genetic Vectors, Membrane Transport Proteins physiology, Microscopy, Confocal, Oligopeptides metabolism, Protein Transport physiology, Recombinant Fusion Proteins metabolism, Trabecular Meshwork cytology, Anterior Eye Segment metabolism, Gene Products, tat metabolism, Profilins metabolism, Trabecular Meshwork metabolism
Abstract

Purpose: Fusion proteins containing a protein transduction domain (PTD4) are able to cross biological membranes. We tested the applicability of the protein transduction method for study of the aqueous humor trabecular outflow pathway by targeting the actin cytoskeleton, which is known to be involved in outflow facility regulation., Methods: Expression vectors useful for generating fusion proteins with the PTD4 domain and the actin-binding protein Profilin I were constructed. The transductional and functional properties of these proteins were tested in bovine trabecular meshwork cells in culture. The effects of PTD4-Profilin I on outflow facility were evaluated in perfused bovine anterior segments. PTD4-beta-galactosidase was used to visually check correct delivery of fusion proteins to trabecular meshwork cells., Results: The fusion proteins generated were characterized by western blot. Immunocytochemistry experiments showed intracellular staining for PTD4-Profilin I in trabecular meshwork cells in culture. The fusion protein was found in the cytoplasm associated with actin filaments and in the leading edge of the cellular membrane. In contrast, control Profilin I, without the PTD4 domain, was unable to cross the cell membrane. In perfused anterior segments, 2 microM PTD4-Profilin I increased trabecular outflow facility in a reversible manner, while Profilin I had no significant effect. Anterior segments perfused with PTD4-beta-galactosidase showed positive staining in the trabecular meshwork tissue., Conclusions: Protein transduction technology is a valuable tool for targeting trabecular meshwork tissue, not only for performing physiological studies, but also as a potential drug-delivery method. Profilin I action on the actin cytoskeleton further reinforces the importance of this structure in outflow facility regulation.

Published
2005

2. Identification of Kir2.1 channel activity in cultured trabecular meshwork cells

Author

Llobet, A., Gasull, X., Pales, J., Eulalia Marti, and Gual, A.

Subjects
Patch-Clamp Techniques, Potassium Channels, Dose-Response Relationship, Drug, Blotting, Western, 8-Bromo Cyclic Adenosine Monophosphate, Cesium, Barium, Trabecular Meshwork, Potassium, Potassium Channel Blockers, Animals, Humans, Calcium, Cattle, Potassium Channels, Inwardly Rectifying, Cells, Cultured
Abstract

To study the presence of inwardly rectifying K(+) (Kir) channels in cultured bovine (BTM) and human (HTM) trabecular meshwork cells.Cultures of BTM and HTM cells were obtained by an extracellular matrix digestion technique. Whole-cell patch-clamp recordings of BTM cells were performed with the appropriate solutions to detect K(+) currents. Also, Western blot analysis of Kir2.1 protein expression was performed on both cultured BTM and HTM cells.A strong inwardly rectifying current at negative potentials to the equilibrium potential for K(+) (E(K+)) and highly selective for K(+) was detected in 60% of cultured BTM cells. The slope conductance of the inward rectification was more pronounced when the extracellular [K(+)] was increased and was proportional to [K(+)](0.45). The current was blocked by Ba(2+) and Cs(+) in a voltage- and concentration-dependent manner, with K(d) at 0 mV, of 74.7 microM and 45.6 mM, respectively. Current amplitude was reduced by increasing extracellular [Ca(2+)]. The current was insensitive to 10 microM glibenclamide and 10 nM tertiapin. The application of 100 microM 8-Br-cAMP reduced the current by 50%. Kir2.1 channel expression was detected in confluent monolayers of BTM and HTM cells by Western blot analysis.A population of cultured BTM cells expressed an inwardly rectifying K(+) current that illustrates the biophysical and pharmacologic characteristics of the detected Kir2.1 channel protein. Kir2.1 channels are also thought to be present in HTM cells. Kir2.1 channels could be related to TM physiology, because they are involved in contractile and cell volume regulatory responses, two mechanisms that modify TM permeability.

Published
2001

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