Ellen B. Wolpert

Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood–brain or blood–retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65‐ or 2.57‐fold increase in content, respectively. A similar two‐fold increase in occludin mRNA was observed by real‐time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO‐1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.

The testis isoform of the phosphorylase kinase catalytic subunit (PhK-T) plays a critical role in regulation of glycogen mobilization in developing lung

In order to identify the form of phosphorylase kinase catalytic subunit expressed in developing lung, degenerate polymerase chain reaction primers were designed based on conserved domains of the two known catalytic subunits, expressed primarily in muscle and testis. Amplification of cDNA from day 19 fetal rat lung followed by cloning and sequence analyses indicated that only the testis isoform of phosphorylase kinase (PhK-T) was detectable in fetal lung. In situ hybridization analyses indicated that expression of PhK-T RNA in developing lung tissue was widespread and not restricted to Type II epithelial cells; PhK-T protein expression was temporally and spatially correlated with expression of PhK-T RNA. PhK-T RNA and protein expression was also characterized in the PhK-deficient glycogen storage disease (gsd) rat. PhK-T RNA levels were similar in Type II cells isolated from wild type and gsd/gsd fetuses; in contrast, PhK-T protein was virtually undetectable in gsd/gsd Type II cells and enzyme activity was very low. These results suggest that PhK-T plays a critical role in mobilization of glycogen during fetal lung development and that failure to catabolize glycogen in the gsd/gsd rat is related to an untranslatable PhK-T RNA or unstable protein.