Calcium uptake and calcium-dependent phosphorylation during development of rat brain neurons in culture

The emergence of the capacities for calcium uptake and calcium-regulated protein phosphorylation during the development of embryonic brain neurons in tissue culture was examined. In the maturing cells, the enhancement in ⁴⁵Ca²⁺-uptake upon stimulation with high K⁺ increased by 3-4 fold during the second week in vitro, in parallel to an increase in the capacity for high K⁺-induced Ca²⁺-dependent release of prelabeled [³H]dopamine. The pattern of incorporation of [³²P_i]phosphate into the major phosphoproteins in maturing cells under nonstimulating conditions also changed during cell development: the incorporation of ³²P_i into two proteins of apparent molecular weights - 55,000 and 43,000 dalton - increased, but decreased in a 45,000 dalton protein. Stimulation of mature cells (after 10-11 days in vitro) resulted in a Ca²⁺-dependent increase in the amount of ³²P_i incorporated into the 43,000 dalton protein and a decrease in the amount incorporated into the 55,000 dalton protein. This calcium-regulated phosphorylation pattern was not observed until 6 days in vitro. Introduction of Ca²⁺ into the immature cells by means of the Ca²⁺ ionophore A23187 did not alter the phosphorylation pattern and did not cause neurotransmitter release. The amount of [³⁵S]methionine incorporated into a 43,000 dalton protein which comigrated with the 43,000 dalton phosphoprotein also increased upon cell maturation. The results suggest that this phosphoprotein (which does not comigrate with nonphosphorylated actin on two-dimensional polyacrylamide gels) develops in the cells in parallel to the emerging processes of the stimulation-induced calcium entry and calcium-dependent neurosecretion.