Rod photoreceptor disc shedding in eye cups: relationship to bicarbonate and amino acids

We previously demonstrated that disc shedding in eye cups from Xenopus laevis depends on medium bicarbonate concentration, but also found that other unidentified medium components influenced the response. Here, in order to determine which medium components influence shedding, we used high bicarbonate salt solutions supplemented with medium components in amphibian tissue culture medium. Compared to the high bicarbonate salt solution alone, addition of fetal bovine serum or whole egg ultrafiltrate enhanced disc shedding. However, a response fully comparable to that in intact animals was obtained on addition of a mixture of 14 amino acids. The full light-evoked response depended on the simultaneous presence of high bicarbonate (35 mM) and amino acids. In the presence of amino acids, photoreceptor tips and RPE showed tight interdigitation and less tendency for in vitro separation, suggesting that these medium components promote such interdigitation. Using the defined medium we have additionally shown that shedding is related to HCO3- concentration under conditions of controlled medium pH, Cl- content and osmolality.