Source: UTAH STATE UNIVERSITY submitted to
REGULATION OF INTESTINAL PHOSPHATE ABSORPTION
 
PROJECT DIRECTOR: Nemere, I.
 
PERFORMING ORGANIZATION
NUTRITION & FOOD SCIENCE
UTAH STATE UNIVERSITY
LOGAN,UT 84322
 
NON TECHNICAL SUMMARY: Certain agricultural practices contribute to the problem of phosphorus in water. The poultry industry is constrained by the need to feed this indispensible nutrient, but plagued by the presence of the unabsorbed portion in manure. This project examines the mechanisms of phosphate absorption in chicken intestine, especially as stimulated by a hormonally active form of vitamin D. Some aspects of the proposal include the effect of age, the role of a novel hormone binding protein (receptor), and a particular signaling pathway that appears to be specific for phosphate absorption.
 
OBJECTIVES: The goal of the proposed research is to further our understanding of the molecular and cellular mechanisms that govern the absorption of phosphate, a micronutrient central to structural, metabolic, and regulatory processes. Preliminary experiments have indicated that enterocytes from young chicks can be stimulated to rapidly take up phosphate through a protein kinase C (PKC)-dependent mechanism that can be activated by 1,25(OH)2D3 binding to a membrane associated, rapid response steroid (1,25D3-MARRS) protein binding (bp). Both phosphate uptake in cells and PKC decline with age, while the binding affinity of the 1,25D3-MARRS bp decreases with age. Further investigation will include (1) Verification that 1,25(OH)2D3-stimulated phosphate uptake in isolated intestinal epithelial cells of chickens, and changes with age, accurately reflects transport; (2) Evaluation of the role of the 1,25D3-MARRS bp in the rapid uptake of phosphate. This specific aim has three sub-aims (2a) Assessing mRNA levels for the protein. This will establish whether changes in responsiveness to steroid are due to decreased transcription. (2b) Loss of function studies. This will quite definitively establish whether or not the 1,25D3-MARRS bp is the membrane 'receptor' for the steroid. (2c) Gain of function studies. This will determine if the robust uptake phenotype can be rescued. (2d) Analyses of protein metabolism and interactions. These studies will determine whether expression of 1,25D3-MARRS bp or a binding partner, decreases with age. (3) Further delineation of the role of PKC in mediating phosphate transport. By defining the molecular and cellular mechanisms that regulate phosphate transport, new methods may become apparent for enhancing absorption, and decreasing the manure content of phosphorous in poultry. This will contribute to the sustainability of US agriculture.
 
APPROACH: 1. To study transport, duodenal loops from 7-wk, 14-wk, 28-wk, and 58 wk old birds will be luminally perfused with 32P and vascularly perfused with either control media or media containing 130 pM 1,25(OH)2D3. Determination of radioactivity in the venous effluent will indicate transport. 2a. mRNA levels for the 1,25D3-MARRS bp will be assessed as follows: mucosal scrapings from the same age groups listed in aim (1) will be snap frozen in liquid nitrogen. Total RNA will be prepared with the Trizol reagent, level and purity determined by spectrophotometry, and 1 ug of each prepartion used for reverse transcriptase PCR. Products will be resolved on agarose gels and stained with ethidium bromide to allow for densitometry. 2b. Intestinal epithelial cells will be isolated by chelation, and plated overnight in RPMI 1640. The following morning the cells will either be untreated, or transfected with control or active ribozyme (against MARRS bp) with liptofectamine. After further incubation, cells will be harvested to asses 32P uptake, specific binding of [3H]1,25(OH)2D3, PKC activation and MARRS bp levels. 2c. Gain of function studies will be conducted with an expression vector containing the cDNA for the MARRS bp. The same parameters, as in loss of function studies, will be assessed. 2d. Cell cultures prepared from intestinal epithelium of appropriate aged birds will be incubated with 35S-amino acids to allow labeling. Homogenates will then be incubated with immobilzied Ab099 to the MARRS bp. After exhaustive washing, bound proteins will be eluted with acidic pH, resolved on SDS-PAGE, and exposed to x-ray film. Age-associated changes in band intensities will be analyzed densitometrically. 3. Initial studies will use a pan-PKC to determine steroid-mediated redistribution to membranes as judged by Western analyses and confocal microscopy. After optimizing incubation times with isolated intestinal cells, equivalent experiments will be performed with commercially available antibodies to the various PKC isotypes.
 
CRIS NUMBER: 0198249 SUBFILE: CRIS
PROJECT NUMBER: UTA00240 SPONSOR AGENCY: CSREES
PROJECT TYPE: NRI COMPETITIVE GRANT PROJECT STATUS: TERMINATED MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Dec 1, 2003 TERMINATION DATE: Nov 30, 2007

GRANT PROGRAM: IMPROVING ANIMAL GROWTH AND DEVELOPMENT
GRANT PROGRAM AREA: Animal Systems

CLASSIFICATION
Knowledge Area (KA)Subject (S)Science (F)Objective (G)Percent
133321010106.120%
133322010106.120%
302321010102.210%
302321010302.210%
302321010402.210%
302322010102.210%
302322010302.210%
302322010402.210%

CLASSIFICATION HEADINGS
KA302 - Nutrient Utilization in Animals
KA133 - Pollution Prevention and Mitigation
S3220 - Meat-type chicken, live animal
S3210 - Egg-type chicken, live animal
F1030 - Cellular biology
F1010 - Nutrition and metabolism
F1040 - Molecular biology
G2.2 - Increase Efficiency of Production and Marketing Systems
G6.1 - Ensure Clean Water and Air


RESEARCH EFFORT CATEGORIES
BASIC 100%
APPLIED (N/A)%
DEVELOPMENTAL (N/A)%

KEYWORDS: chickens; vitamin d; phosphates; intestines; nutrient uptake; nutrient transport; plasma membranes; receptors; protein kinase; metabolic regulation; pollution control; nutrient utilization; molecular biology; cell biology; broilers; layers; micronutrients; animal nutrition; mechanism of action; enzyme activity; protein binding; epithelial cells; verification; metabolic pathways; age; signal transduction

PROGRESS: Dec 1, 2003 TO Nov 30, 2007
OUTPUTS: Protein kinase C (PKC) is involved in both the rapid 1,25(OH)2D3-mediated uptake of phosphate1 and extrusion of calcium2 in chick intestinal epithelial cells. In this study we assessed the contribution of various isotypes. Using Western analyses we found that a 1-min treatment of isolated enterocytes resulted in dose-dependent increases in PKCa and PKCB in subsequently prepared P2 fractions (post nuclear pellet containing intracellular organelles and basal lateral membranes), but not P1 fractions. Anti-PKCa staining increased in P2 fractions after treatment of cells with 300 pM steroid and declined at 650 pM hormone, relative to corresponding controls, while anti PKCB immunoreactivity was elevated in P2 fractions prepared from cells treated with either 300 pM or 650 pM 1,25(OH)2D3. Thus PKCa redistribution corresponded to the dose-response curve for phosphate uptake and PKCB redistribution corresponded to the dose-response curve for calcium efflux. We further verified the role of PKCa in hormone stimulated phosphate uptake by transfecting primary cultures of intestinal cells with siRNA for that isotype. Using confocal microscopy it was necessary to reduce hormone exposure to 30 sec in order to view redistribution. Omission of primary antibody as a staining control revealed nonspecific fluorescence in all regions of the cell. Using average pixel intensity to correct for this, PKCa was found to increase significantly in the lateral apical membrane after a 30 sec exposure of cells to 300- or 650 pM 1,25(OH)2D3 (P < 0.01 and 0.001, respectively). By comparison, anti-PKCB immunofluorescence was found to increase significantly (P < 0.01) in the basal region of cells, relative to controls, following exposure of cells to 300 pM seco-steroid. PARTICIPANTS: Utah State University and USDA TARGET AUDIENCES: Poultry Science Departments, Biomedical community. Results from this projected have been presented at national meetings of the Endocrine Society, Society for Bone and Mineral Research, and Experimental Biology

IMPACT: 2003-12-01 TO 2007-11-30 These findings further delineate the moledcular entities involved in mediating hormone-stimulated phosphate uptake or calcium efflux. Defects in mineral absorption from the intestine may in the future be traced to decreased function of these pathways.

PUBLICATION INFORMATION: 2003-12-01 TO 2007-11-30
Nemere, I, Hintze, K 2007. Novel Hormone 'Receptors'. J Cellular Biochem :.

PROJECT CONTACT INFORMATION
NAME: Nemere, I.
PHONE: 435-797-3286
FAX: 435-797-2379