Demonstration of growth hormone receptors in cultured rat epiphyseal chondrocytes by specific binding of growth hormone and immunohistochemistry

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Abstract
Growth hormone has been reported to exert direct effects on rat and rabbit epiphyseal chondrocytes in vitro, indicating that GH interacts with specific receptors on these cells. To investigate this possibility, binding of GH to cultured rat epiphyseal chondrocytes was studied under various experimental conditions. Chondrocytes were isolated enzymatically from epiphyseal growth plates of the proximal tibia of 20-day-old male rats and were cultured in monolayer in Ham's F-12 medium supplemented with 10% calf serum and 1% of a serum substitute. The cells were seeded at various densities (25 000–200 000 cells/well) and cultured for 5–16 days. Twenty-four hours before binding experiments, the medium containing calf serum was changed for one containing serum obtained from hypophysectomized rats, in order to avoid binding of GH present in the calf serum. Binding was studied by incubating 125I-labelled human GH (hGH) with the cells in the presence or absence of various concentrations of unlabelled hGH, bovine GH (bGH), rat GH (rGH) and ovine prolactin (oPRL). Specific binding could be demonstrated in cells cultured for 5–16 days. Binding was dependent upon time and temperature, and maximal binding was obtained by incubating the labelled hormone for 4–6 h at 24 °C. An increase in binding was noted between 7 and 12 days in culture. In cells cultured for 12 days, addition of unlabelled hGH, bGH or rGH caused a dose-dependent displacement of 125I-labelled hGH, whereas oPRL was ineffective. Scatchard analysis resulted in a linear plot, and the number of binding sites/cell was approximately 5700, with a dissociation constant of 0·46 nmol/l. The increase in binding between days 7 and 12 was independent of the density of seeded cells, but total binding was higher if the cells were seeded at a low density. By using a monoclonal antibody to the rabbit GH receptor, specific staining could be demonstrated immunohistochemically in the cultured cells. The results show the presence of GH receptors in cultured rat epiphyseal chondrocytes and also show that the culture conditions influence the expression of GH receptors. Journal of Endocrinology (1989) 122, 69–77