Effect of FGFs on Adult Bovine Muller Cells: Proliferation, Binding and Internalization

Abstract

A new method for culturing retinal Muller cells from adult bovine tissue is described. The identification of these glial cells was based on immunocytochemical analysis of specific Muller cell markers. Cultured cells from fourth to ninth passage showed positive labelling for S 100 protein, carbonic anydrase (CAA), glutamine synthetase (GS), α cristallin (αC) and polyclonal glial fibrillary acidic protein (GFAP) antibody, but were negative for both monoclonal GFAP antibody and also for Muller cells in the retina. Investigation of the effect of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and epithelial growth factor (EGF) on the proliferation of the Muller cells revealed that bFGF was the most potent mitogen (EC₅₀ = 14 pM). Binding data revealed the presence of two classes of binding sites for aFGF and bFGF: (1) a high affinity binding site (Kd of 14 pM and 27 pM for aFGF and bFGF respectively); (2) a low affinity binding site (Kd of 3.2 nM and 0.6 nM for aFGF and bFGF respectively with great variability in the number of binding sites). In addition, the cross-linking experiments revealed the presence of high molecular weight FGF receptors (110-140 kDa). After aFGF or bFGF binding to Muller cells, aFGF and bFGF-cell surface receptors were rapidly downregulated with a half-life for disappearance of 35-50 min. Internalization and degradation of ¹²⁵I-bFGF bound to the Muller cell receptors did not occur at 4°C. At 37°C, however, there was a rapid decrease in receptor-bound ¹²⁵I-bFGF due to the downregulation of bFGF rjeceptors. Concomitantly ¹²⁵T-bFGF appeared inside the Muller cells. After 2 h, ¹²⁵I-bFGF began to be degraded and after 6 h three fragments of 16 kDa, 8 kDa and 5.5 kDa were discernible. Degradation of bFGF appeared to occur in the lysosomal compartment since it was inhibited by chloroquine, an inhibitor of lysosomal proteases; aFGF internalization and degradation followed the same kinetics as bFGF with the appearance of 7 kDa and 5 kDa fragments. These results suggest that Muller cells may be the target for aFGF and bFGF contained in other cells of the retina. The fact that aFGF could be released from rod outer segment by a phosphorylation-dependent mechanism, and that apical prolongation of the Muller cells is connected with the photoreceptor cells suggest that these factors may be the mediators involved in the communication between glial cells and neurons.

Key Words:

• aFGF
• bFGFm Muller cells
• immunocytochemical analysis