The long-term objective of this proposal is to improve our understanding of the mechanisms whereby hormones and ions control the renal biosynthesis of 1,25(OH)2D3. Because 1,25(OH)2D3 controls net calcium and phosphorus absorption by the intestine, increased skeletal requirements for these elements result in enhanced 1,25(OH)2D3 production. The enzyme that hydroxylates 25-hydroxyvitamin D3 to form 1,25(OH)2D3 is located in the mitochondria of kidney proximal tubules. Parathyroid hormone (PH) and low serum phosphorus are the two major stimuli for 25-hydroxy-1-hydroxylase (1OHase) activity, and blood ionized calcium modulates the response to PTH. Using well established techniques to measure in vitro 1,25(OH)2D3 production rates by proximal tubules prepared from vitamin D-replete rats, studies are proposed to explore the role of cAMP and cytosolic free calcium as intracellular messengers that regulate mitochondrial 1OHase activity during PTH stimulation, low calcium and low phosphorus diet. Cytosolic calcium will be measured by standard fluorescent dye methodology, and intracellular calcium levels will be raised or lowered by agents that bind calcium or release calcium from intracellular stores. The role of cAMP will be investigated by measuring tubule cAMP and the response of 1OHase activity to added dibutryl cAMP. Because PTH stimulation of the 1OHase may be modulated by both blood ionized calcium (suppression) and insulin (stimulation), the effect of extracellular Ca++ and insulin on cAMP formation, cytosolic calcium and intracellular calcium will be determined. Also, the interaction of insulin and PTH will be explored in vitro by determining the effects of insulin on 1OHase activity in tubules from rats fed low calcium, or low phosphorus, or normal mineral diets. Because of the pivotal role of 1,25(OH)2D3 in the control of blood ionized calcium and the delivery of calcium and phosphorus for skeletal mineralization, disturbances in the renal synthesis of this steroid hormone can result in bone disease, bone loss, urolithiasis, renal impairment and soft tissue calcification. Prevention and treatment of these conditions will be more rational if the fundamental disturbances are known in detail.