Role of Phosphodiesterase 4 Isoenzyme in Alkaline Phosphatase Activation by Calcitonin in Porcine Kidney LLC-PK₁ Cells

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To confirm the intracellular signal transduction in regulation of alkaline phosphatase (ALP) activity by calcitonin in kidney tubular cells, effects of several inhibitors of cyclic nucleotide phosphodiesterase (PDE) isoenzymes and cyclic AMP-dependent protein kinase (PKA) on the action of salmon calcitonin in porcine kidney tubular epithelial cells LLC-PK₁ were examined. A confluent culture of LLC-PK₁ cells was treated with calcitonin and inhibitors in Dulbecco’s modified Eagle’s medium supplemented with 0.1% bovine serum albumin, and intracellular cyclic AMP content and ALP activity were measured after incubation for 30 min and 48 hr, respectively. Calcitonin and PDE 4 inhibitors increased cyclic AMP level and ALP activity in the cells, and PDE 4 inhibitors synergistically potentiated the effects of calcitonin. Calcitonin induced ALP activation by treatment for the first 1 hr, as well as continuous treatment for 48 hr, while it never increased the enzyme activity just after 1-hr exposure. Rolipram, an inhibitor of PDE 4 isoenzyme, induced ALP activation by itself and in combination with calcitonin by only a long term treatment (48 hr). The activation of ALP by calcitonin and rolipram each alone and in combination was completely abolished by a PKA inhibitor, H-89. These results confirm that calcitonin induces ALP activation through the cyclic AMP-PKA pathway and that PDE 4 isoenzyme is closely associated with the calcitonin-receptor system and plays a major role in hydrolysis of cyclic AMP produced in the kidney tubular cells.