Ken-ichi Miyamoto (1), Ryoji Suzuki (1), Takashi Horita (2,#), Shinya
Yamamoto (1), Yoshihiro Waki (1) and Kenzo Takagi (3)
(1) Division of Pharmacy and Health Sciences, Graduate School of Natural
Science and Technology, Kanazawa University, 13-1 Takara-machi, Kanazawa
920, Japan
(2) Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3 Kanagawa-machi, Kanazawa 920-11, Japan
(3) Department of Internal Medicine, Nagoya University School of Medicine,
65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan
(#) Present address: Tatsumi Kagaku Co., Ltd., 3-345 Minma, Kanazawa 921,
Japan
Abstract: 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.
Keywords: Calcitonin, Alkaline phosphatase, Phosphodiesterase 4, Kidney,
LLC-PK1 cell