Abstract
Abstract: :
Purpose: We have previously shown that lactose and structurally related sugars support the assembly of nascent photoreceptor outer segments in the absence of the retinal pigment epithelium (RPE). The purpose of the present study was to determine if lactose-mediated support of photoreceptor outer segment assembly demonstrates characteristics of a receptor-mediated phenomenon. Methods: The RPE was removed from isolated Xenopus laevis embryonic eyes that were allowed to complete differentiation in: (1) Niu-Twitty medium; (2) Niu-Twitty medium with concentrations of lactose or isopropyl beta-D-thiogalactopyranoside (IPTG) over four orders of magnitude; or (3) Niu-Twitty medium with lactose or IPTG for 2 days after which medium were changed to Niu-Twitty alone. Control retinas matured in vitro with an adherent RPE. Retinal morphology was evaluated for organized folding of outer segment membranes. As a first step to identify the putative lactose receptor, extracted retinal total proteins from stage 33/34 Xenopus laevis tadpoles were applied to an N-acetyllactosamine affinity column. After extensive washing of the column, proteins were eluted with 30mM galactose followed by electrophoretic separation and silver staining. Results: IPTG, a non-metabolizable form of galactose, promoted the formation of organized outer segment assembly similar to lactose. IPTG and lactose supported outer segment assembly in a step-wise fashion with maximal support at 0.05mM and 5mM, respectively. Removal of lactose or IPTG resulted in loss of support of outer segment assembly. By affinity chromotography, three proteins (apparent molecular weights of 175, 67 and 56 kD) were eluted from the column by 30mm galactose. Conclusion:Lactose and IPTG support photoreceptor outer segment assembly via a non-metabolic pathway, this effect is reversible and maximal at given concentration. These data suggest that lactose support of photoreceptor outer segment assembly demonstrates characteristics of a receptor-mediated phenomenon. The putative "lactose receptor" is likely one of the three proteins which were isolated by affinity chromotography. A molecular characterization of these proteins is pending.
Keywords: 564 retinal development • 541 receptors: pharmacology/physiology • 560 retinal culture