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

Abstract

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 45Ca 2+-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 Ca 2+-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 Ca 2+-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 Ca 2+ into the immature cells by means of the Ca 2+ 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.