Inhibition of retinal growth cone activity by specific metalloproteinase inhibitors in vitro

The developing neural retina expresses a set of extracellular proteases including plasminogen activator and gelatinases. Since neurites of retina cells cultured on fluorescent gelatin digest the substrate in their paths, we have suggested that the proteases are used by the tips of growing fibers to allow them to migrate within the mass of the tissue in vivo. In order to obtain further information about relationships between extracellular proteases and fiber growth, we have examined the effects of the specific inhibitors HS-LFA (HS-Leu-Phenylala-Ala, enantiomeric forms 1 and 2), bathophenanthroline sulfonate (BPS), phenylmethyl sulfonyl fluoride (PMSF), and relevant controls on the activity of retinal growth cones in vitro, monitored by time lapse video microscopy. Of the inhibitors tested, only the two enantiomeric forms of HS-LFA caused a reproducible cessation of both spike extension and filopodial processes at the growth cone ruffling, while control media had no effect. In some cases, the growth cone swelled and exhibited small protrusions. The behavior of growth cones was in sharp distinction to that of the cytoplasm of neural cells, and membrane ruffling of flat cells, which continued in activity throughout. Growth cone activity returned after several hours in the presence of the agent. BPS was toxic at concentrations above 2.5 mM. Below that, it had no effect. L-cysteine, PMSF, and control media had no effect. The relevance of these results to the possible role of proteases in fiber outgrowth from retinal cells is discussed.