Sodium ion-dependent (N-methylamino)-alpha-isobutyric [correction of isobutryic] acid uptake by embryonic chick cells exposed to ethanol in ovo:response to the stimulation/downregulation of protein kinases.

Abstract

Amino acid uptake, critical for embryonic development, was studied in cultured chick cells isolated from 5-day-old chick embryos that had received treatment in ovo with either vehicle (sterile chick Ringer's solution) or vehicle plus ethanol (1.5 g/kg). Upon being placed in culture, the cells were grown in the absence of ethanol per se. The uptake of (N-methylamino)-alpha-isobutyric [correction of isobutryic] acid (AIB) was used to estimate amino acid uptake via system A, a Na+-dependent system that transports short-chain amino acids. The uptake of AIB in cultured chick embryo cells in response to the stimulation/downregulation of three protein kinases [tyrosine kinase, cyclic AMP-dependent kinase A (PKA) and protein kinase C (PKC)] was determined. Acute activation of PKC by treatment of the cells with a phorbol ester (TPA) inhibited Na+-dependent AIB uptake. Conversely, treatment of the cells with TPA for 24 h, to downregulate PKC activity, significantly increased AIB uptake. The data suggest that in these cells, PKC plays an important role in the regulation of amino acid uptake via system A. Activation of PKA via treatment of the cells with forskolin, an adenylyl cyclase activator, had little effect on AIB uptake in cells from vehicle-treated embryos and only a slight depressing effect on uptake by cells from ethanol-treated embryos. Insulin and insulin-like growth factor-1 (IGF-1) both stimulated AIB uptake, but the higher concentrations of insulin necessary to increase uptake to a level comparable to that of IGF-1 stimulation suggested that insulin could be acting through the IGF-1 receptor. Thus, while AIB uptake by cells of in ovo ethanol-treated embryos was significantly increased by treatment with either insulin or IGF-1, IGF-1 appeared to be the more physiologically important compound in the chick embryo. Overall, there was a consistent trend for cells isolated from in ovo ethanol-treated embryos to have higher levels of AIB uptake relative to cells isolated from vehicle-treated embryos, regardless of the in vitro treatment.