Mitochondrial alkaline phosphatase as an intracellular signal in the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 in LLC-PK1 cells.

In previous works we have found a mitochondrial alkaline phosphatase (AP) activity in LLC-PK1. The aim of this work has been to study the possible involvement of mitochondrial AP activity in the synthesis of 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3) and 24,25-dihydroxyvitamin D3 (24,25(OH)(2)D-3) from the substrate 25(OH)D-3. Renal phenotype LLC-PK1 cells were incubated with 25(OH)D-3 as substrate and treated with or without 1,25(OH)(2)D-3, forskolin, 12-myristate-13-acetate (PMA) and 1,25(OH)(2)D-3 in conjunction with PMA. Incubation of LLC-PK1 cells with forskolin (adenylate cyclase activator) not only stimulated the 1-hydroxylase and inhibited the 24-hydroxylase activities but also increased the mitochondrial AP activity. The addition of 1,25(OH)(2)D-3, the main activator of 24-hydroxylase, produced a decrease of mitochondrial AP activity, a decrease of 1,25(OH)(2)D-3 synthesis and an increase of the 24,25(OH)(2)D-3 synthesis. Incubation with PMA, a potent activator of protein kinase C, did not produce any changes in mitochondrial AP activity, but an inhibition of 1,25(OH)(2)D-3 and an activation of 24,25(OH)(2)D-3 synthesis were found. Moreover, incubation of LLC-PK1 cells with PMA in conjunction with 1,25(OH)(2)D-3 produced an additive effect in the decrease of 1,25(OH)(2)D-3 and an increase of 24,25(OH)(2)D-3 synthesis remaining mitochondrial AP activity as cells treated only with 1,25(OH)(2)D-3. Our results suggest that mitochondrial AP activity could be involved as an intracellular signal in the regulation of 25(OH)D-3 metabolism to the synthesis of 1,25(OH)(2)D-3 and 24,25(OH)(2)D-3 in renal phenotype LLC-PK1 cells through cAMP protein kinase system.