The Effect of a Somatostatin Analog on the Transepithelial Transport of ARPE 19 Cells

Abstract

Purpose: : Somatostatin (SST) analogues such as octreotide have been used to improve visual acuity and reduce macular edema in proliferative diabetic retinopathy but the mechanism is unclear. SST is known to regulate tight junction integrity and ion currents in neurons. Since RPE cells, which form the outer blood retinal barrier, express SST receptors 1, 2 and 5 we asked whether SST analogues modulate transepithelial transport by the intercellular (modulation of tight junctions) and/or by the intracellular (ion channel) routes in RPE cells.

Methods: : The osmotic challenge assay was used to determine fluid flow across tight monolayers of ARPE19 cells grown in transwell inserts. Octreotide at 0–300µM was added to either the upper or lower well and fluid flow measured over a 3 hr period. Intracellular transpithelial transport was evaluated by measuring changes in intracellular Ca ²⁺ in ARPE cell monolayers following addition of octreotide at 1, 10, and 100 µM over a 30 minute period using FURA–2 real–time imaging and the FLIPR calcium flux assay. The FLIPR membrane potential assay was used to detect changes in membrane potential due to ion flux across the RPE plasma membrane following octreotide treatment. The effect of octreotide on occludin and phospho–occludin expression was determined by Western Blot analysis.

Results: : Octreotide demonstrated a dose–dependent increase in fluid flow by 30 minutes across the RPE with maximal flow reached at 10µM. This response was sustained over a period of three hours. Real time imaging and the calcium flux assay showed that octreotide caused a significant increase in intracellular Ca ²⁺ at all concentrations tested at 30 minutes consistent with an increase in epithelial permeability. Octreotide was able to modulate ion flux across the RPE membrane resulting in membrane hyperpolarization. Surprisingly, Western blot analysis demonstrated an increase in the tight junction protein occludin after treatment with octreotide.

Conclusions: : Taken together these data confirm that SST analogs act on both intercellular and intracellular RPE transepithelial transport and suggest a role for SST analogs in improving BRB function and RPE function in diabetic macular edema. SST analogs must elicit this effect by cell–cell adhesion molecules other than occludin.

Keywords: drug toxicity/drug effects • pharmacology • retinal pigment epithelium