Date of Award:

2004

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Nutrition, Dietetics, and Food Sciences

Department name when degree awarded

Nutrition and Food Sciences

Advisor/Chair:

Ilka Nemere

Abstract

The effects of 1,25(OH)2D3, PTH and 25(OH)D3 on phosphate or calcium uptake were studied in cultured, adherent chick enterocytes over a period of 10 min after hormone addition. Time course studies of cells treated with 130 pM 1,25(OH)2D3 showed an increase in 32P uptake as early as 3 min. Similar studies with 65 pM bPTH(l1-34) resulted in an increase in 45Ca uptake only if the cells had been cultured in serum. (OH)D3, which is not firmly established as an active metabolite of vitamin D, was shown to increase 45Ca uptake within 5 min at a 100 nM concentration.

Analyses of signal transduction events involving each hormone were undertaken using PKC and PKA inhibitors, chelerythrine and Rp-cAMP, respectively. In the presence of PKC inhibitor and 1,25(OH)2D3 elevated 32P levels were apparent; however, further investigations involving efflux studies showed PKC inhibition of 32P extrusion in the presence or absence of hormone. On the other hand, suppression of the PKA pathway stimulated an increase in 1,25(OH)2D3-mediated 32P uptake. Preincubation of enterocytes with Ab099 against a putative membrane receptor for 1,25(OH)2D3 abolished steroid-stimulated 32P uptake.

While PKC inhibition had no effect on 45Ca uptake in enterocytes exposed to 65 pM bPTH(1-34) in serum, pretreatment with PKA inhibitor resulted in 45Ca levels relatively close to basal levels. Cells pretreated with PKC inhibitor and exposed to 25(OH)D3 demonstrated no changes in 45Ca levels, whereas inhibition of PKA induced decreased 45Ca levels after 10 min of incubation.

In equivalent time course studies of membrane trafficking using confocal microscopy, potential vectorial transport initiated by each hormone was analyzed with agonist alone or in the presence of PKC and PKA inhibitors. In addition 1,25(OH)2D3 was tested in the presence of Ab099 against its putative membrane receptor. Visualization of these experiments using the endocytotic marker dye, FM 1-43, demonstrated that hormone-mediated membrane trafficking is rapid enough to contribute to ion transport. These results also suggest that vectorial vesicular transport mechanisms were involved to some extent in response to each hormone. Moreover, the pattern for membrane trafficking was different for each agonist.

These combined results indicate that adherent chick enterocytes demonstrate hormone-mediated uptake that occurs more rapidly than cells in suspension or in perfusion studies. This research supports previous studies that identify 25(OH)D3 as an active vitamin D metabolite. The PKA signal transduction pathway is a possible mechanism for PTH- and 25(OH)D3-mediated increases in 45Ca. In addition, a central role for the basal lateral membrane receptor protein, 1,25(OH)2D3MARRS-bp, in 1,25(OH)2D3-mediated 32P uptake is supported. Confocal imaging suggests that the transport mechanism for phosphate or calcium ions in the presence of these hormones involves vesicular carriers.

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