25-Hydroxyvitamin D3 metabolism in mitochondria from primary renal cultures

Previous in vitro studies concerning the renal metabolism of 25-hydroxyvitamin D3 (25(OH)D3) to form 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and 24,25R-dihydroxyvitamin D3 (24,25(OH)2D3) have utilized intact cell systems. In reflecting upon the possible mechanisms by which hormonally induced changes in the production of 1,25(OH)2D3 and 24,25(OH)2D3 may be brought about, we asked whether altered mitochondrial hydroxylase activities can quantitatively account for changes in the total cellular output of these steroids. Our objective was to delineate between extramitochondrial processes (e.g. altered substrate delivery), and those events restricted to the renal mitochondria (altered hydroxylase activities). We have examined the effect of pretreating primary cultures of chick kidney cells with either 1,25(OH)2D3 or parathyroid hormone (PTH) on 25(OH)D3-hydroxylase activities present in subsequently isolated mitochondria. Pretreatment with 10(-7) M 1,25(OH)2D3 reduced 1 alpha-hydroxylase activity in both cells and mitochondria to approximately 60% of control values by 1 h, and to 25-30% by 2 h. The effect of PTH (10 ng/ml) in both mitochondrial and whole cell preparations was an approximate 40% increase in measured 1 alpha-hydroxylase activity. 10 microM forskolin (FSK) elicited an approximate 2-fold increase in 1,25(OH)2D3 production. Reciprocal effects were observed with respect to 24-hydroxylase activity in both whole cell and mitochondrial preparations in response to exogenous 1,25(OH)2D3, PTH, and FSK. The findings demonstrate that these hormones initiate intracellular events which lead directly to altered 25(OH)D3 1 alpha- and 24-hydroxylase activities within the renal mitochondria.