IN-SITU BINDING AND IMMUNOCYTOCHEMISTRY OF INSULIN-LIKE GROWTH-FACTOR-I RECEPTORS IN PRIMARY CULTURES OF PORCINE ADIPOSE-TISSUE STROMAL-VASCULAR CELLS TREATED WITH INDOMETHACIN

An autoradiographic technique for localizing [I-125]IGF-I binding was adapted to primary cultures of pig stromal-vascular (S-V) cells to study the effects of pig serum (PS) and (or) indomethacin (Indo) on IGF-I and fibronectin (FN) receptors. Computer assisted image analysis revealed that the percentage of [I-125]IGF-I binding decreased (P < .001) in cultures (7 d) treated with Indo compared with 2% PS alone. Binding of [I-125]IGF-I analog (low-binding affinity for the IGF-I binding proteins) also decreased (P < .001) in 7-d cultures treated with Indo. The unlabeled IGF-I analog was more effective than unlabeled IGF-I at decreasing (P < .001) binding of [I-125]IGF-I. Number of fat cell clusters and fat cell area (total lipid deposition) increased (P < .001) with PS + Indo compared with PS or Indo alone. Immunocytochemical staining (fluorescein isothiocyanate) of IGF-I receptors with a polyclonal antibody (flow cytometric analysis) decreased (P < .001) from 1 to d 3 and did not change from 3 to 7 d with PS alone. However, the percentage of reactive cells decreased (P < .05) when Indo was added to PS. In contrast, the number of cells detected with the FN receptor antibody increased fourfold (P < .01) from 1 to 7 d in cultures with PS alone and decreased (P < .05) when Indo was present. Undifferentiated cells around fat cell clusters were reactive for the IGF-I receptor, but fat cells showed no reactivity. These results indicate that Indo decreased total [I-125]IGF-I binding, and flow cytometric analysis verified that Indo down-regulated IGF-I and FN receptors, while increasing differentiation of pig S-V cells in primary cultures.