Receptors for epidermal growth factor (urogastrone) and insulin in primary cultures of rat hepatocytes maintained in serum-free medium

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Abstract
We have analyzed the receptors for epidermal growth factor (urogastrone) (EGF-URO) and insulin in primary cultures of adult rat hepatocytes maintained for up to 3 weeks on human placental cell matrix in serum-free defined medium. Cross-link labeling experiments revealed that the insulin receptor, partially damaged by the collagenase isolation procedure, was rapidly regenerated to yield an intact receptor. In contrast, cross-link labeling of the EGF-URO receptor revealed that, upon prolonged culture, there was a progressive disappearance of the high molecular mass (175 kilodaltons (kDa)) receptor form, and an appearance of low molecular mass receptor species (130 and 105 kDa). After 3 weeks of culture, the low molecular mass receptor forms accounted for all of the labeled EGF-URO receptor present in the cells. Measurements of EGF-URO binding indicated that the number of EGF-URO binding sites per cell (2.0 × 105 ± 0.3 × 105) did not change during the 3 weeks of culture. However, there was a decrease in EGF-URO binding affinity, reflected by an increase in the KD from 0.6 to 3.0 nM. At zero time and after 3 weeks in culture, Scatchard plots of the binding data were linear; at intermediate time points, the plots were curvilinear. Despite the changes in the EGF-URO receptor that occurred, cells were still responsive to EGF-URO in terms of the inhibition of acetate incorporation into lipid. The ED50 for EGF-URO (about 0.2 nM) was the same for short-term cultures (48 h) as for cells maintained in culture for 3 weeks. We conclude that the long-term culture of hepatocytes in serum-free medium yields an altered low molecular form of the EGF-URO receptor that is, nonetheless, functional. The study points to differential changes in receptors for peptide hormones that may occur in long-term hepatocyte cultures and illustrates the feasibility of using such cultures for metabolic studies of the actions of EGF-URO.