The overall goal of this project is to elucidate the function(s) of 1,25-dihydroxyvitamin D3(1,25(OH)2D3), or the lack thereof, in new putative target tissues by addressing the hypothesis that 1,25(OH)2D3, exerts specific and important effects therein via previously described receptors. Thus, 1,25(OH)2D3 function will be rigorously evaluated by a series of physiological and biochemical criteria in the model systems rat heart and testis. The first phases of these studies are focused on testing the hypothesis that 1,25(OH)2D3 plays a role in intracellular calcium homeostasis. However, because of the fallacy in limiting such studies to any one area of possible functions, the hypothesis that 1,25(OH)2D3 affects these systems through alternate mechanisms must be tested in the future. Whether there is a correlation between vitamin D status or 1,25(OH)2D3 receptor and physiological function will be tested by evaluating the effects of vitamin D status in normocalcemic rats on the contractile properties of heart muscles and on testicular function and examining whether 1,25(OH)2D3 receptor levels in putative new target tissues correlate with physiological status of the vitamin D endocrine system. Effects of 1,25(OH)2D3 status and receptors on intracellular calcium homeostasis in heart and Sertoli cells will be evaluated by studying the hormonal effects on uptake of 45Ca into intact cultured heart myocytes and Sertoli cells. Effects of 1,25(OH)2D3 on selected mechanisms/subcellular organelles will be evaluated by determining 45Ca uptake in heart myocytes and Sertoli cells in the absence and presence of pharmacological probes. Additional studies will extend studies of the effects of 1,25(OH)2D3 status/receptors on calcium binding proteins detected by 45CA binding to proteins transblotted to nitrocellulose membranes, on proteins modulated by calmodulin by the 125-I-calmodulin gel overlay technique, and by immunocytochemical localization of calcium binding proteins that demonstrate vitamin D regulation. These interrelated studies of 1,25(OH)2D3 function and receptors in putative new targets will add much to our understanding of the vitamin D endocrine system and will provide a basis for future assessments of 1,25(OH)2D3 function in both normal and diseased individuals.
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