Thomas Yorio

The Role of Glia, Mitochondria, and the Immune System in Glaucoma

Author(s): Tezel, Gulgun; Fourth ARVO/Pfizer Ophthalmics Research Institute Conference Working Group

Sorbinil prevents the hypergalactosemic-induced reduction in [3H]-myo-inositol uptake and decreased [3H]-myo-inositol incorporation into the phosphoinositide cycle in bovine lens epithelial cells in vitro.

The synthesis of phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4-5-bisphosphate was studied using 3H-myo-inositol (3H-MI) as precursor in cultured bovine lens epithelial cells (BLECs) maintained in galactose-free, physiological medium or 40 mM galactose (Gal) +/- sorbinil for six days. The formation of inositol polyphosphates from phosphoinositides was also shown. Galactitol did not exceed 2mM in Gal-incubated cells after six days of exposure; no galactitol was observed in BLECs maintained in galactose-free, physiological medium or Gal supplemented with sorbinil. Uptake of 3H-myo-inositol(3H-MI) into BLECs was significantly reduced in cells exposed to Gal. A concomitant reduction in 3H-MI incorporation was observed in the phosphoinositides, as well as with the released inositol phosphates. The simultaneous addition of sorbinil to the Gal medium corrected the drop in 3H-MI uptake and normalized 3H-MI incorporation into the phosphoinositides and inositol phosphates. While an apparent decrease in the three inositol-containing lipids was observed with the Gal-incubated cells, based on 3H-MI incorporation, there was no change in total membrane phosphatidylinositol content when compared to cells maintained in physiological medium as determined by the microgram Pl PO4 per microgram total membrane PO4. The apparent loss of radiolabeled phosphoinositides was attributed to the decreased specific activity resulting from the lower internal pool of 3H-MI in the Gal-exposed cells available for incorporation into the phosphoinositides.(ABSTRACT TRUNCATED AT 250 WORDS)

Uncoupling of Attenuated myo-[3H]Inositol Uptake and Dysfunction in Na+-K+-ATPase Pumping Activity in Hypergalactosemic Cultured Bovine Lens Epithelial Cells

Attenuation of both the active transport of myo-inositol and Na+-K+-ATPase pumping activity has been implicated in the onset of sugar cataract and other diabetic complications in cell culture and animal models of the disease. Cultured bovine lens epithelial cells (BLECs) maintained in galactose-free Eagles minimal essential medium (MEM) or 40 mM galactose with and without sorbinil for up to 5 days were examined to determine the temporal effects of hypergalactosemia on Na+-K+-ATPase and myo-inositol uptake. The Na+-K+-ATPase pumping activity after 5 days of continuous exposure to galactose did not change, as demonstrated by 86Rb uptake. The uptake of myo-[3H]inositol was lowered after 20 h of incubation in galactose and remained significantly below that of the control throughout the 5-day exposure period. The coadministration of sorbinil to the galactose medium normalized the myo-[3H]inositol uptake. No significant difference in the rates of passive efflux of myo-[3H]inositol or 86Rb from preloaded galactose-treated and control cultures was observed. Culture-media reversal studies were also carried out to determine whether the galactose-induced dysfunction in myo-inositol uptake could be corrected. BLECs were incubated in galactose for 5 days, then changed to galactose-free physiological medium with and without sorbinil for a 1-day recovery period. myo-Inositol uptake was reduced to 34% of control after 6 days of continuous exposure to galactose. Within 24 h of media reversal, myo-inositol uptake returned to or exceeded control values in BLECs switched to either MEM or MEM with sorbinil. These observations support the contention that the aldose reductase reaction or formation of the product of the aldose reductase reaction disrupted lens cell myo-inositol uptake. Although the coadministration of sorbinil to the galactose medium protected against the attendant decrease in transport activity, the cessation of exposure of BLECs to galactose was sufficient stimulus to normalize myo-inositol uptake. Moreover, these observations suggested that the deficit in myoinositol uptake elicited by exposure to galactose was reversible and occurred independently of changes in Na+-K+-ATPase pumping activity in cultured lens epithelium, indicating that the two parameters are not strictly associated and that the deficit in myo-inositol uptake occurs rapidly during hypergalactosemia.

DEPLETION OF GLUTATHIONE BY L-BUTHIONINE SULFOXIMINE DOES NOT PROMOTE INACTIVATION OF MYO-INOSITOL TRANSPORT IN CULTURED BOVINE LENS EPITHELIAL CELLS

Exposure of cultured bovine lens epithelial cells (BLECs) to minimal essential medium (MEM) containing 40 mM galactose (Gal) promotes a decrease in the cellular content of reduced glutathione (GSH) as galactitol increases. Incubation of BLECs with Gal also leads to a reduction in 3H-myo-inositol (3H-MI) concentrating capability. Studies were therefore initiated to determine the nature of the relationship between polyol accumulation, GSH content and the attenuation of the active transport of myo-inositol. The inhibitor of glutathione biosynthesis, L-buthionine sulfoximine (L-BSO) was used in order to lower the intracellular pool of GSH in MEM-maintained cells to a concentration below that characteristically observed in Gal-treated cells, under conditions whereby no galactitol accumulation could or had occurred. L-BSO (0.5 mM) was simultaneously administered to BLECs maintained in either MEM or Gal for up to five days. The cellular content of GSH after five days of continuous incubation was 3.3 micrograms GSH/micrograms PO4 in MEM alone and 0.45 microgram GSH/microgram PO4 in MEM + BSO. Moreover, the GSH content in BLECs exposed to Gal for five days was 1.9 micrograms GSH/micrograms PO4 and was not detectable in the Gal + BSO-treated cells. However, the ability to concentrate 3H-MI in MEM + BSO-treated BLECs was equivalent to that observed with MEM-maintained cells regardless of the significant difference in GSH content. Likewise, L-BSO addition to Gal-treated cells, while virtually depleting the intracellular GSH content, did not further decrease the ability of the cells to accumulate 3H-MI compared to that observed with BLECs in Gal alone. Indeed, supplementation of Gal-treated cells with exogenous GSH failed to correct the Gal-induced attenuation in myo-inositol concentrating ability. These studies demonstrate that the Gal-induced depletion of cellular GSH and the Gal-induced deficit in ability to concentrate myo-inositol are not associated and represent independent events. That is, depletion of lens cell GSH does not lead to the attenuation of myo-inositol uptake in cultured lens epithelial cells.

Protein kinase C activity and its relationship to myo-inositol uptake during hyperglycemic conditions in cultured bovine lens epithelial cells

Incubation of cultured bovine lens epithelial cells (BLECs) in minimal essential medium (MEM) containing 40 mM galactose for 20 hr results in an attenuation of 3H-myo-inositol (3H-MI) concentrating ability. Decreased MI uptake could negatively impact on normal phosphoinositide turnover and diacylglycerol production, and presumably, protein kinase C (PKC) activation. The present report examines the relationship between PKC activity, myo-inositol transport and hyperglycemic conditions. PKC activities in the cytosol and particulate fractions of bovine lens epithelial cells in culture were quantitated using a mixed micelle assay following DEAE-cellulose (DE52) and Sephadex G-25 chromatography. Protein kinase C activity was assessed as Ca2+ and phospholipid-dependent Ac-myelin basic protein substrate peptide phosphorylation and confirmed using a PKC pseudosubstrate inhibitor peptide (PKC 19-36). Total PKC activity was similar in galactose-incubated cells (871 +/- 64 pmol/mg total protein/min) and control cells (881 +/- 8 pmol/mg total protein/min) after 20 hr. In unstimulated cells, approximately 90% of the total cellular PKC activity was recovered in the cytosolic fraction. Enzyme translocation was induced with the tumor promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), resulting in a 6-fold increase in membrane-associated PKC activity. A similar PMA-induced translocation was observed in BLECs incubated with 40 mM galactose MEM-maintained cells briefly treated with PMA or the non-phorbol PKC activators, SC-10 and mezerein, displayed a rate of 3H-MI uptake similar to the untreated control cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The Eye as a Drug Target

Publisher Summary Blindness affects millions of people worldwide and results not only from genetic problems at birth and accidents, but also from the chronic debilitating diseases, such as macular degeneration, glaucoma and cataracts. As people age, the eye diseases become more prevalent and have a major impact on the quality of life for most of the people. Thus, the eye has become an important target for drug development to provide relief and protection from a variety of eye diseases. The eye has special characteristics that make it an excellent target for selective drug delivery and for ease in monitoring the effectiveness of new therapies. This includes a number of tissues that are transparent so as to allow for the transmission of light to the retina. These tissues include the cornea and lens. This characteristic also allows for the ease of monitoring to gauge what happens at the retina, and to be able to access various compartments of the eye. Although the eye has two blood-ocular barriers and is an immune privileged site, it is still one of the most accessible organs of the body which can be reached with rather local drug administration, including the ability to apply drugs topically.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.