Abstract
The steroid hormone 1,25‐dihydroxyvitamin D~3~ [1,25(OH)~2~D~3~] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25‐dihydroxyvitamin D~3~ [24,25(OH)~2~D~3~] inhibits the rapid stimulation by 1,25(OH)~2~D~3~. Earlier work in this laboratory has indicated that a cellular binding protein for 24,25(OH)~2~D~3~ is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H~2~O~2~ production, studies were undertaken to determine if pro‐oxidant conditions mimicked the inhibitory actions of 24,25(OH)~2~D~3~, and anti‐oxidant conditions prevented the inhibitory actions of 24,25(OH)~2~D~3~. An antibody against the 24,25(OH)~2~D~3~ binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)~2~D~3~‐mediated ^32^P uptake. Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)~2~D~3~, each in the presence of the catalase inhibitor 3‐amino‐1,2,4‐triazole, or with 1,25(OH)~2~D~3~ alone. Cells exposed to hormone alone again showed an increased accumulation of ^32^P, while cells treated with catalase inhibitor and hormone had uptake levels that were indistinguishable from controls. We tested whether inactivation of protein kinase C (PKC), the signaling pathway for ^32^P uptake, occurred. Incubation of cells with phorbol‐13‐myristate (PMA) increased ^32^P uptake, while cells pretreated with 50 µM H~2~O~2~ prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity while cells exposed to H~2~O~2~ prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones. J. Cell. Biochem. 101:1176–1184, 2007. © 2007 Wiley‐Liss, Inc.