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Insulin-like growth factor I (IGF-I) is thought to stimulate bone resorption indirectly through a primary effect on osteoblasts, which in turn activate osteoclasts by as-yet-unidentified mechanisms. Small decreases in extracellular pH (pHo) dramatically increase the resorptive activity of osteoclasts. Our purpose was to characterize the effect of IGF-I on acid production by osteoblastic cells. When confluent, UMR-106 osteoblast-like cells and rat calvarial cells acidified the compartment beneath them. Superfusion with IGF-I caused a further decrease in pHo. To investigate the mechanism, we monitored acid efflux from subconfluent cultures. IGF-I rapidly increased net efflux of H+ equivalents in a concentration-dependent manner. IGF-II (10 nM) evoked a smaller response than IGF-I (10 nM). The response to IGF-I was partially dependent on extracellular Na+, but not glucose, and exhibited little if any desensitization. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, abolished the response to IGF-I but not to parathyroid hormone. Thus IGF-I enhances acid efflux from osteoblastic cells, via a signaling pathway dependent on activation of phosphatidylinositol 3-kinase. In vivo, acidification of the compartment between the osteogenic cell layer and the bone matrix may affect diverse processes, including mineralization and osteoclastic bone resorption.

The ability to manipulate genetic information in the germ line of mice has provided powerful approaches to study gene function in vivo. These approaches have included the establishment of mouse lines in which a specified gene or genes are overexpressed, ectopically expressed, or deleted. Transgenic and gene-targeted mouse lines have been used extensively to study the function of the insulin-like growth factors (IGF), IGF-I and IGF-II, and their receptors and binding proteins. In the IGF system, these technologies have elucidated the roles of the IGFs in fetal and somatic growth and have demonstrated a critical role for this system in transformation and tumorigenesis. Analysis of combinatorial crosses of gene-targeted mouse lines also has suggested the existence of an as yet unidentified IGF receptor that regulates fetal growth. Similar approaches using transgenic and gene-targeted mouse models have been initiated to study the in vivo functions of the IGF binding proteins. These mouse models provide important tools to test specific functional questions in vivo as well as to study the long-term physiological consequences of chronic gene alterations.

We previously showed that insulin and insulin-like growth factor (IGF)-I and IGF-II caused a dose-dependent increase in permeability through the paracellular pathway of T84 cell monolayers over 3-4 days. Here we have determined which cell surface receptors were involved in this response. Using radioligand binding studies and receptor cross-linking studies, we found that T84 cells possess insulin and IGF-I receptors. There were approximately 20 x 10(3) insulin receptors/cell with a dissociation constant (KD) of 0.5 nM and 29 x 10(3) IGF-I receptors with a KD of 0.6 nM for IGF-I. Cross-linking studies identified the alpha-subunit of insulin and IGF-I receptors with deduced molecular weights of 126 x 10(3) and 128 x 10(3), respectively. IGF-II bound to T84 cells with an apparent KD of approximately 2.0 nM. Radioreceptor cross-linking indicated that IGF-II interacted principally with the IGF-I receptor, although low levels of the IGF-II/mannose 6-phosphate receptor were also expressed on the cell surface. We then correlated the biological effect with the radioligand binding studies. It was first demonstrated that insulin and IGF-I were degraded in medium in the presence of cells. In addition, we showed that continuous exposure for 2-3 days to insulin or IGF-I was required to produce their biological effect on permeability. Taking into account the rate of degradation and the requirement for continuous exposure, we found a close correlation between radioligand binding and the half-maximal effective concentration for the hormonal effects on transepithelial permeability.(ABSTRACT TRUNCATED AT 250 WORDS)