Ontogeny of the L-type voltage sensitive calcium channels in chick embryo retinospheroids

The L-type voltage sensitive calcium channels (VSCC) of chick embryo retinospheroids were characterized during the development in vitro. Functionally, the activity of VSCC was characterized by continuously monitoring the changes in the intracellular free Ca2+ concentration (delta[Ca2+]i) with indo-1, in response to 30 mM KCl. The contribution of the L-type VSCC was evaluated using the L-type VSCC antagonist, nitrendipine. We also characterized the binding of [3H]nitrendipine to retinospheroid membranes during development, and determined the Kd and Bmax values. We observed that the changes in [Ca2+]i in response to 30 mM KCl increased from 159.46 +/- 6.62 nM at 0 days in vitro (DIV) retinospheroids to 704.4 +/- 59.9 nM at 14 DIV retinospheroids. Nitrendipine (2 microM) blocked the delta[Ca2+]i response by approximately 67% in all ages tested. No significant difference in the Kd values for the nitrendipine binding was observed during in vitro development of the retinospheroids. However, the Bmax increased from 27.99 +/- 1.95 fmol/mg protein in 0 DIV retinospheroids to 131.09 +/- 14.24 fmol/mg protein in 14 DIV retinospheroids, supporting the delta[Ca2+]i results. The results presented suggest that the increase in [Ca2+]i during development was due to an increase in the number of L-type channels. Therefore, the expression of L-type VSCC is developmentally regulated during retinogenesis in vitro and accompanies neuronal maturation, probably regulating the Ca2+ input crucial to the onset of important intracellular Ca2+-dependent functions.