THE EFFECT OF INSULIN AND INSULIN-LIKE GROWTH-FACTOR-I ON THE EXPRESSION OF CALRETININ AND CALBINDIN D-28K IN RAT EMBRYONIC NEURONS IN CULTURE

In a primary culture of rat embryonic neurons, insulin (5 mg/ml) promoted neurite formation between neuron clusters in serum/glial-free simplified medium with a high concentration of transferrin (100 pg/ml). Insulin growth factor-1 (IGF-1) exerted a similar effect in the same culture system at a lower dose (100 ng/ml) of insulin. Calretinin has recently been identified as a calcium binding protein expressed predominantly in sensory neurons, which has six calcium binding domains and shows molecular similarity to calbindin D-28k, an intestinal calcium transporter protein also found in the CNS. The effects of insulin and IGF-1 on the expression of calretinin and calbindin D-28k were investigated in rat embryonic neuronal cell culture. When calls were cultured for 2 days, insulin and ICF-1 promoted the expression of both proteins; when cultured for more than 2 days, IGF-1 still exerted a growth factor effect, but insulin decreased the expression of calretinin. Using the present culture system, we demonstrated that the effects of closely related molecules, insulin and ICF-1 which share receptors reciprocally, differed in calretinin and calbindin D-28k expression. Therefore, these two calcium binding proteins may play different physiological roles in the nervous system based either on different molecular mechanisms or switching roles al different stages of CNS development. Amplification based on Western blot employing the streptoavidin-horseradish peroxidase method was applied to detect calretinin and calbindin D-28k in the culture system by an immunoblotting technique.