Newborn Rabbit Gastric Smooth Muscle Cell Culture

Abstract

SummaryEpidermal growth factor (EGF) and transforming growth factor‐alpha (TGF‐α) are two in a family of growth‐promoting peptides for many gastrointestinal epithelia. This study was designed to assess their mitogenic effect on cultured gastric myocytes and to characterize specific EGF receptors on these cells. Single myocytes were isolated from newborn rabbit gastric fundus and placed into tissue culture. The composition of the culture at confluence as assessed by immunostaining with smooth muscle actin‐specific monoclonal antibody (CGA7) was >95% myocytes. To assess the effect of putative growth factors, freshly isolated myocytes were incubated in Dulbecco's modified Eagle's (DME) medium containing 1% fetal bovine serum in the presence or absence of growth factors. After 6 days, cells were incubated in serum‐free medium with [3H]thymidine (1 μCi/ml) in the continued presence or absence of growth factors. After 24 h, EGF and TGF‐α but not insulin‐like growth factor I (IGF‐1) induced a dose‐dependent increase in [3H]thymidine incorporation. Ten nanomolar EGF or TGF‐α increased [3H]thymidine incorporation more than sixfold over control. EGF was more potent than was TGF‐α, with apparent median effective dose (ED50) values of 64 ± 14 pM and 166 ± 62 pM (p < 0.05), respectively. EGF bound to cultured myocytes with Kd = 7.6 ± 1.8 nM and Bmax = 27 ± 11 pmol/mg DNA or 440,000 receptors/cell. TGF‐α competed for binding at these receptors. Although IGF‐I did not stimulate thymidine incorporation, specific high‐affinity receptors for IGF‐I were detected on gastric myocytes. These results suggest that the EGF family may play a role in the growth of gastric smooth muscle.