1,25(OH)2D3-mediated phosphate uptake in isolated chick intestinal cells:: Effect of 24,25(OH)2D3, signal transduction activators, and age

We have previously reported an absence of a 1,25(OH)(2)D-3-mediated effect on Ca-45 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)(2)D-3 increased P-32 uptake within 5 min of addition, and reached 150% of controls after 10 min (P<0.05). Both isolated enterocyles and the perfused duodenal loop system exhibited apparent biphasic dose-response curves for 1,25(OH)(2)D-3-stimulated P-32 uptake and transport, and inhibition of stimulation by 24,25(OH)(2)D-3. 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 P-32 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)(2)D-3 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)(2)D-3 had no effect on enterocytes from 14 or 28 wk birds. Western analyses with Ab 099 against the 1,25(OH)(2)D-3 (1,25D(3))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)(2)D-3 activation. In enterocytes from 7-14- or 28-week birds, PKC was enhanced 170, 120, and 105% of controls, respectively. The combined data validate P-32 uptake in isolated enterocytes as a model system to study 1,25D(3)-MARRS protein function, and indicate that for phosphate transport, the rapid actions of 1,25(OH)(2)D-3 are physiologically more important in growing animals than immature ones.