1,25(OH)2D3-mediated phosphate uptake in isolated chick intestinal cells: effect of 24,25(OH)2D3, signal transduction...

We have previously reported an absence of a 1,25(OH)2D3-mediated effect on 45Ca handling by intestinal epithelial cells isolated from normal chicks (Nemere and Campbell [2000] Steroids 65:451-457). In the current work, we provide evidence that in similar cell preparations, 1,25(OH)2D3 increased 32P uptake within 5 min of addition, and reached 150% of controls after 10 min (P < 0.05). Both isolated enterocytes and the perfused duodenal loop system exhibited apparent biphasic dose-response curves for 1,25(OH)2D3-stimulated 32P uptake and transport, and inhibition of stimulation by 24,25(OH)2D3. A comparison of signal transduction activators demonstrated the following parallels in both isolated intestinal cells and perfused duodena: lack of effect of forskolin (a protein kinase (PK) A activator) on 32P handling, but simulation by BAY K8644 (a calcium channel activator) and phorbol ester (a PKC activator). Finally, we tested the effect of 1,25(OH)2D3 on phosphate uptake in epithelial cells isolated from birds of increasing ages (7, 14, and 28 wk). In contrast to the robust response of cells from young, growing chicks, 1,25(OH)2D3 had no effect on enterocytes from 14 or 28 wk birds. Western analyses with Ab 099 against the 1,25(OH)2D3 (1,25D3)-Membrane-Associated Rapid Response Steroid (MARRS) binding protein revealed a decrease in average density of the immunoreactive band with age. PKC activity determined in isolated epithelial cells exhibited a decrease in average basal (control) activity with age, as well as a decrease in response to 1,25(OH)2D3 activation. In enterocytes from 7-14- or 28-week birds, PKC was enhanced 170, 120, and 105% of controls, respectively. The combined data validate 32P uptake in isolated enterocytes as a model system to study 1,25D3-MARRS protein function, and indicate that for phosphate transport, the rapid actions of 1,25(OH)2D3 are physiologically more important in growing animals than immature ones.