Rapid desensitization of parathyroid hormone dependent adenylate cyclase in perifused human osteosarcoma cells (SaOS-2).
The pulsatile but not the continuous application of parathyroid hormone (PTH) increase bone mass in vivo. To study the effects of intermittent hormonal administration on bone-derived cells in vitro, we established a perifusion system using the human osteosarcoma cell line SaOS-2. Cells were grown in suspension culture attached to collagen beads and were then loaded into a 3 ml syringe for perifusion experiments. The application of PTH(1-34) resulted in a dose-dependent increase of cAMP release by SaOS-2 cells into the effluent medium. Cyclic AMP accumulation was rapidly desensitized by approx. 80% after 30 min of continuous exposure to PTH(1-34) (10(-7) M), while cells remained responsive to forskolin. The recovery of PTH responsiveness required at least 2 h of hormone-free perifusion. Desensitization in the experimental setting was dose-dependent (EC50 = 1 x 10(-10) M PTH(1-34)). Neither 8Br-cAMP (2 x 10(-4) M) nor PMA(1 x 10(-7) M) had an effect on the PTH(1-34)-induced desensitization of the adenylate cyclase. Radioreceptor assays showed that [125I]-[Tyr36]hPTHrP(1-36)amide binding to SaOS-2 cells was decreased by 60-70% by PTH(1-34) (1 x 10(-6) M), bPTH(1-84) (1.8 x 10(-6) M) and bPTH(3-34) (2 x 10(-6) M), whereas 8Br-cAMP (2 x 10(-4) M) had no effect on radioligand binding. PMA (1 x 10(-7) M) appeared to slightly increase [125I]PTHrP binding. This observation is consistent with a small (3-fold) increase in PTH-induced cAMP release as a result of PMA pre-treatment. Receptor internalization was dose-dependent EC50 = 3 x 10(-7) M PTH(1-34)). The maximal effect occurred after 10-30 min and was largely reversible within 2 h. Monensin (3 x 10(-5) M) inhibited the recovery from receptor internalization. We conclude that a perifusion system using SaOS-2 cells is a suitable model to study the effect of discontinuous application of PTH on cAMP release. A rapid, homologous desensitization of PTH(1-34) stimulated cAMP accumulation has been observed that does not appear to involve protein kinase A or C.
Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston 02114.
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Bergwitz
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Abou-Samra
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Hesch
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Jüppner
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000242:Cyclic AMP / Q000032:analysis; D006801:Humans; D012516:Osteosarcoma / Q000201:enzymology*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D010477:Perfusion / Q000379:methods; D018016:Receptors, Parathyroid Hormone / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D010281:Parathyroid Hormone; D018016:Receptors, Parathyroid Hormone; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D013755:Tetradecanoylphorbol Acetate