The major goal of this research project is to elucidate intracellular signal transduction pathways by which metabolic changes modulate and hormones regulate the Na+-Pi symport across brush border membranes (BBM). The studies are based on the following hypothesis. While hormones such as PTH or dopamine regulate Na+-Pi symport via cAMP-PKA and/or pathway(s), the change in metabolic states namely gluconeogenesis (GNG) of proximal tubular (PT) modulate the Na+ Pi symport via a novel pathway involving NAD+, cyclic adenosine diphosphoribose (CADPR), and Ca2+ release. The final steps of the cADPR signalling pathway is regulation of microtubule (MT) disassembly and assembly by microtubule associated proteins (MAP2), which are phosphorylated by Ca2+ dependent and cAMP- dependent protein kinases. Specific objective include: 1. To determine the characteristics of cADPR system in PT cells and OK cells. This includes subcellular localization and enzymatic properties of ADP-cyclase, cADRP-glucohydrolase (cADPR-GH), and dynamics of cellular cADPR levels. 2. To examine binding of cADPR onto specific cADPR-receptor and its characteristic (KD, Bmax, specificity, pH optimum, etc) and relationship between cADPR binding and release of Ca2+ from the intracellular Ca2+ stores. To determine whether multifunctional Ca2+/CaM protein kinases-II (Ca/CaM-PK-II) is activated by Ca2+ released in response to cADPR, and whether it phosphorylates microtubule associated proteins (MAP2), and whether phosphorylated MAP2 promote disassembly of MT in PT cells. Further, to test whether PKA also phosphorylates MAP2 and whether the effects of both protein kinases are additive. 3. To determine whether the cADPR signalling system operates in intact cells as integral pathway and modulates Na+-Pi symport across BBM in response to change in PT metabolism, namely GNG. 4. To delineate intracellular mechanisms by mutual """"""""crosstalk"""""""" between the hormonal signals acting via cAMP-PKA pathway and metabolic signals acting via cADPR-Ca2+ pathway in PT cells. We will also test the hypothesis which states that the degree of MT assembly in cytoplasm of PT cells determines the extent of internalization and reinsertion of Na+- Pi symporters into BBM, and thereby regulates the Na+-Pi reabsorption. We propose that MAP2 phosphorylation by the two kinases is the focal point of additivity or synergism.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK030759-14
Application #
2138507
Study Section
General Medicine B Study Section (GMB)
Project Start
1982-04-01
Project End
1998-03-31
Budget Start
1995-04-01
Budget End
1996-03-31
Support Year
14
Fiscal Year
1995
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
de Toledo, F G; Cheng, J; Liang, M et al. (2000) ADP-Ribosyl cyclase in rat vascular smooth muscle cells: properties and regulation. Circ Res 86:1153-9
Liang, M; Chini, E N; Cheng, J et al. (1999) Synthesis of NAADP and cADPR in mitochondria. Arch Biochem Biophys 371:317-25
Caride, A J; Chini, E N; Penniston, J T et al. (1999) Selective decrease of mRNAs encoding plasma membrane calcium pump isoforms 2 and 3 in rat kidney. Kidney Int 56:1818-25
de Toledo, F G; Thompson, M A; Bolliger, C et al. (1999) gamma-L-glutamyl-L-DOPA inhibits Na(+)-phosphate cotransport across renal brush border membranes and increases renal excretion of phosphate. Kidney Int 55:1832-42
Caride, A J; Chini, E N; Homma, S et al. (1998) mRNAs coding for the calcium-sensing receptor along the rat nephron: effect of a low-phosphate diet. Kidney Blood Press Res 21:305-9
Chini, E N; de Toledo, F G; Thompson, M A et al. (1997) Effect of estrogen upon cyclic ADP ribose metabolism: beta-estradiol stimulates ADP ribosyl cyclase in rat uterus. Proc Natl Acad Sci U S A 94:5872-6
de Toledo, F G; Beers, K W; Berndt, T J et al. (1997) Opposite paracrine effects of 5-HT and dopamine on Na(+)-Pi cotransport in opossum kidney cells. Kidney Int 52:152-6
de Toledo, F G; Cheng, J; Dousa, T P (1997) Retinoic acid and triiodothyronine stimulate ADP-ribosyl cyclase activity in rat vascular smooth muscle cells. Biochem Biophys Res Commun 238:847-50
Beers, K W; Thompson, M A; Chini, E N et al. (1996) beta-Estradiol inhibits Na+-P(i) cotransport across renal brush border membranes from ovarectomized rats. Biochem Biophys Res Commun 221:442-5
Beers, K W; Chini, E N; Dousa, T P (1995) All-trans-retinoic acid stimulates synthesis of cyclic ADP-ribose in renal LLC-PK1 cells. J Clin Invest 95:2385-90

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