DIFFUSIBLE, RETINAL FACTORS STIMULATE THE BARRIER PROPERTIES OF JUNCTIONAL COMPLEXES IN THE RETINAL-PIGMENT EPITHELIUM

The retinal pigment epithelium lies at the interface between the neura l retina and the choriocapillaris where it forms a blood-retinal barri er. Barrier function requires a polarized distribution of plasma membr ane proteins and 'tight' tight junctions. During chicken embryogenesis , these features develop gradually. Although terminal junctional compl exes are established by embryonic day 4, the distribution of the Na+/K +-ATPase is not polarized in all cells of the epithelium until embryon ic day 11. Similarly, the tight junctions of early embryos are leaky, but become tight by hatching (embryonic day 21). We used primary cell culture to examine the molecular basis of this gradual induction of po larized function. Pigment epithelium harvested from embryonic day 7, a nd cultured on filters, formed monolayers coupled by junctional comple xes. The distribution of the Na+/K+-ATPase was non-polarized and the t ight junctions were leaky with a transepithelial electrical resistance of 20-30 OMEGA cm2. To isolate diffusible factors that stimulate the transepithelial electrical resistance, neural retinas from embryonic d ay 7, 14 or 16 embryos were incubated at 37-degrees-C in base medium f or 6 hours. The conditioned medium was added to the apical chamber of freshly cultured pigment epithelium. The distribution of the Na+/K+-AT Pase became basolateral, and the electrical resistance gradually incre ased two to three times over 6 days. The increase in electrical resist ance corresponded to a decrease in the rate of [H-3]inulin diffusion a cross the monolayer. The effectiveness of the conditioned medium incre ased steadily with increasing age of the neural retina. Rather than in creased production of an active factor, apparently different active fa ctors were produced at different ages. The embryonic day 14 retinal-de rived factors were heat- and alkali-labile and smaller than 10 kDa. Ad ditionally, they were acid-stable and insensitive to proteolysis by ca thepsin D. These studies provide evidence that diffusible factors from the neural retina regulate the gradual development of barrier functio n in the retinal pigment epithelium.