The present research plan proposes to establish an experimental basis, to examine the direct and specific influence of adrenergic agents in vitro, known to induce changes in eye pressure in vivo, in the target cells, namely the ciliary epithelium, the tissue responsible for aqueous humor formation. Preliminary studies on an NEI Pilot Project award have demonstrated the feasibility of separating PE/NPE in culture. Using human and rabbit eyes of different ages and, if possible, of glaucoma tissues, it is intended to accomplish the following: (1) Enzymatic dissociation and further separation of NPE from PE cells by isopycnic centrifugation. (2) Growth optimization of NPE and PE in hormonally defined culture media. (3) Cellular cloning of NPE and PE cell. (4) Studies on transforming NPE. Screening, isolation and characterization of NPE transformed cells. (5) Criteria to define a number of morphological and biochemical markers to identify NPE and PE cells from other cell types in culture. (6) To investigate biochemical responses of NPE cells, namely protein phosphorylation and secretion, to external stimuli (beta-adrenergic agonists, blockers and cAMP analogs), since Preliminary Studies have shown that a small number of proteins are highly phophorylated in PE cells. This hormone-dependent activation can be antagonized by blocking agents such as Timolol of Propanolol. (7) Identification of the protein substrates highly phosphorylated under hormonal action, by two-dimensional gel electrophoresis and tryptic mapping analysis. Elucidation of the regulatory mechanisms of aqueous humor secretion should be very important in understanding more the functions of the cells involved and should help in the development of pharmacologic means for controlling aqueous humor formation.
| Aroca-Aguilar, Jose-Daniel; Sanchez-Sanchez, Francisco; Martinez-Redondo, Francisco et al. (2008) Heterozygous expression of myocilin glaucoma mutants increases secretion of the mutant forms and reduces extracellular processed myocilin. Mol Vis 14:2097-108 |
| Lopez-Martinez, Francisco; Lopez-Garrido, Maria-Pilar; Sanchez-Sanchez, Francisco et al. (2007) Role of MYOC and OPTN sequence variations in Spanish patients with primary open-angle glaucoma. Mol Vis 13:862-72 |
| Coca-Prados, Miguel; Escribano, Julio (2007) New perspectives in aqueous humor secretion and in glaucoma: the ciliary body as a multifunctional neuroendocrine gland. Prog Retin Eye Res 26:239-62 |
| Sanchez-Sanchez, Francisco; Martinez-Redondo, Francisco; Aroca-Aguilar, J Daniel et al. (2007) Characterization of the intracellular proteolytic cleavage of myocilin and identification of calpain II as a myocilin-processing protease. J Biol Chem 282:27810-24 |
| Ghosh, Sikha; Choritz, Lars; Geibel, John et al. (2006) Somatostatin modulates PI3K-Akt, eNOS and NHE activity in the ciliary epithelium. Mol Cell Endocrinol 253:63-75 |
| Li, Yanxia; Aroca-Aguilar, J Daniel; Ghosh, Sikha et al. (2006) Interaction of myocilin with the C-terminal region of hevin. Biochem Biophys Res Commun 339:797-804 |
| Lopez-Garrido, Maria-Pilar; Sanchez-Sanchez, Francisco; Lopez-Martinez, Francisco et al. (2006) Heterozygous CYP1B1 gene mutations in Spanish patients with primary open-angle glaucoma. Mol Vis 12:748-55 |
| Salvador-Silva, Mercedes; Ghosh, Sikha; Bertazolli-Filho, Rubens et al. (2005) Retinoid processing proteins in the ocular ciliary epithelium. Mol Vis 11:356-65 |
| Aroca-Aguilar, J Daniel; Sanchez-Sanchez, Francisco; Ghosh, Sikha et al. (2005) Myocilin mutations causing glaucoma inhibit the intracellular endoproteolytic cleavage of myocilin between amino acids Arg226 and Ile227. J Biol Chem 280:21043-51 |
| Fidzinski, Pawel; Salvador-Silva, Mercedes; Choritz, Lars et al. (2004) Inhibition of NHE-1 Na+/H+ exchanger by natriuretic peptides in ocular nonpigmented ciliary epithelium. Am J Physiol Cell Physiol 287:C655-63 |
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