Date of Award:

5-2006

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Committee Chair(s)

Ilka Nemere

Committee

Ilka Nemere

Committee

Deloy Hendricks

Committee

Daren Cornforth

Abstract

The steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] rapidly stimulates the uptake of phosphate in isolated chick intestinal cells, while the steroid 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] inhibits the rapid stimulation by 1,25(OH)2D3. Earlier work in this laboratory has indicated that a cellular binding protein for the 24,25(OH)2D3 is the enzyme catalase. Since binding resulted in decreased catalase activity and increased H2O2 production, studies were undertaken to determine if pro-oxidant conditions mimicked the inhibitory actions of 24,25(OH)2D3, and anti-oxidant conditions prevented the inhibitory actions of 24,25(OH)2D3. An antibody against a putative 24,25(OH)2D3 binding protein was found to neutralize the inhibitory effect of the steroid on 1,25(OH)2D3-mediated 32P uptake (P < 0.05 to 0.001 at T=3-10 min of incubation). Incubation of cells in the presence of 50 nM catalase was also found to alleviate inhibition at T=5-10 min of incubation. In another series of experiments, isolated intestinal epithelial cells were incubated as controls or with 1,25(OH)2D3, each in Cells exposed to hormone alone again showed an increased accumulation of 32P from T=5-10 min, 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 32P uptake, occurred. Incubation of cells with 100 nM phorbol-13-myristate (PMA) increased 32P uptake to 143% of controls, while cells pretreated with 50 μM H2O2 prior to PMA did not exhibit increased uptake. Likewise, PMA significantly increased PKC activity at T=1-3 min (P < 0.05, relative to corresponding controls), while cells exposed to H2O2 prior to PMA did not. It is concluded that catalase has a central role in mediating rapid responses to steroid hormones.

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