Intestinal maturation: Characterization of mitochondrial phosphate transport in the rat and its regulation by l,25-(OH)₂ vitamin D₃

The mitochondria play a major role in the regulation of oxidative phosphorylation within the cell. Despite the fact that the enterocytes receive the majority of absorbed phosphate and their high metabolic turnover rate, the role of the intestinal mitochondria in phosphate transport system during maturation is not known. Therefore, the current studies were designed to characterize phosphate transport by jejunal mitochondria of rats during maturation (suckling, weanling, and adolescent rats). The functional integrity of the intestinal mitochondria of suckling and adolescent rats was determined by oxygen consumption studies demonstrating respiratory control ratios of more than 3 when succinate was used as a test substrate. Phosphate uptake was significantly stimulated by the presence of 3 mM ATP at all age groups studied. Maximal phosphate uptake in the presence of 3 mM ATP and 2 mM succinate was 16.5 ± 1.0, 20.5 ± 1 and 28.7 ± 0.4 nmol/mg protein (mean ± SE) in suckling, weanling, and adolescent rats respectively. ATP-dependent phosphate uptake was inhibited by 80% with 100 nM p-MB. Kinetic parameters for ATP stimulated phosphate uptake at 10 s revealed a Km of 4 ± 0.9, 2.8 ± 0.4, and 0.9 ± 0.1 mM and V_max of 5 ± 0.7, 9.5 ± 1, and 11 ± 0.7 nmol/mg protein per 10 s in suckling, weanling, and adolescent rats, respectively. Phosphate uptake was also stimulated by an inwardly directed pH gradient (pH out < pH inside) compared to no pH gradient condition suggesting the presence of PO₄ ⁻/0H⁻ exchange. The kinetic of the PO₄ ⁻/0H⁻ exchange showed a Km of 1.9 ± 0.4 and 2.2 ± 1 mM and a V_max of 1.3 ± 0.4 and 4.4 ±1.5 nmol/mg protein/10 s for suckling and adolescent rats, respectively. Vitamin D deficiency decreased markedly the V_max and Km of ATP-dependent phosphate uptake in adolescent rats to 1.6 ± 0.2 nmol/mg protein/10 s and 0.2 ± 0.1 mM, respectively. l,25-(OH)2 vitamin D3 administration, 8 h before death increased the V_max to 3.06 ± 0.8 (p < 0.01) without a significant change in the Km. These results indicate that phosphate uptake into the mitochondria occurs by a ATP-dependent and PO₄ ⁻/0H⁻ exchange mechanisms. The ATP-dependent mechanism is regulated by l,25-(OH)2 vitamin D₃.