The long term objective of the research proposal is to improve the understanding of the cellular mechanisms whereby hormones and ions control the biosynthesis of 1,25-dihydroxyvitamin D3[1,25(OH)2D3]. 1,25(OH)2D3 production by mammalian renal proximal tubule cells is tightly regulated by the skeletal requirements for Ca and P. Parathyroid hormone (PTH) and low P diet (LPD) are the two major activators of renal mitochondrial 25-hydroxyvitamin D3-1 alpha hydroxylase (1-OHase) activity, and elevated blood ionized Ca (Ca2+) suppresses PTH-stimulated enzyme activity. PTH activation of the 1-OHase may be mediated by either the cAMP or Ca messenger systems, or both. PTH can activate both signalling systems in proximal tubule cells, however the role of each transducer in regulating 1-OHase or the several other physiologic functions under PTH control in these cells is unknown. Although pharmacologic concentrations of cAMP increases in vitro 1,25(OH)2D3 production and in vivo circulating 1,25(OH)2D3, recent evidence suggests that additional or alternate pathway(s) for activation of 1-OHase may exist including: 1) in cultured rat proximal tubule cells hPTH 1-34 increases 1,25(OH)2D3 production at physiologic concentrations, but pharmacologic concentrations are required to increase cAMP; 2) high cAMP concentrations induced by forskolin suppress 1,25(OH)2D3; and 3) PTH-stimulated 1,25(OH)2D3 production in perifused rat proximal tubules is reproduced by combined Ca ionophore (A23187) and phorbol ester (TPA) without a change in tissue cAMP. The present application proposes the hypothesis that the Ca messenger system is the major transducer of PTH activation of 1-OHase under physiologic conditions. The hypothesis will be tested through the following Specific Aims: 1) to determine whether cAMP production is required for PTH activation of the 1-OHase; 2) to determine whether physiologic concentrations of PTH produce sufficient cAMP to activate cAMP-dependent protein kinase; and 3) to determine whether PTH activation of the Ca messenger system through increased cytosolic Ca2+ and plasma membrane Ca influx and efflux and activation of protein kinase-C are required for activation of 1-OHase. Identification of the temporal events in 1-OHase activation by PTH will provide approaches to investigate the mechanisms whereby elevated blood Ca2+ inhibits 1,25(OH)2D3 production by PTH but not by LPD. The hypothesis that PTH and LPD activate separate 1-OHase located in distinct nephron segments rather than separate pathways within the same cell will be tested by measuring 1-OHase activity in microdissected S1, S2 and S3 segments of proximal tubule.
