Rapid desensitization of parathyroid hormone dependent adenylate cyclase in perifused human osteosarcoma cells (SaOS-2)

Abstract

The pulsatile but not the continuous application of parathyroid hormone (PTH) increases 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 (EC 50 = 1 · 10 −10 M PTH(1–34)). Neither 8Br-cAMP (2 · 10 −4 M) nor PMA (1 · 10 −7 M) had an effect on the PTH(1–34)-induced desensitization of the adenylate cyclase. Radioreceptor assays showed that [ 125I]-[Tyr 36]hPTHrP(1–36)amide binding to SaOS-2 cells was decreased by 60–70% by PTH(1–34) (1 · 10 −6 M), bPTH(1–84) (1.8 · 10 −6 M) and bPTH(3–34) (2 · 10 −6 M), whereas 8Br-cAMP (2 · 10 −4 M) had no effect on radioligand binding. PMA (1 · 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 (EC 50 = 3 · 10 −7 M PTH(1–34)). The maximal effect occurred after 10–30 min and was largely reversible within 2 h. Monensin (3 · 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.