RAPID DESENSITIZATION OF PARATHYROID-HORMONE DEPENDENT ADENYLATE-CYCLASE IN PERIFUSED HUMAN OSTEOSARCOMA CELLS (SAOS-2)

The pulsatile but not the continuous application of parathyroid hormon e (PTH) increases bone mass in vivo. To study the effects of intermitt ent hormonal administration on bone-derived cells in vitro, we establi shed a perifusion system using the human osteosarcoma cell line SaOS-2 . Cells were grown in suspension culture attached to collagen beads an d were then loaded into a 3 ml syringe for perifusion experiments. The application of PTH(1-34) resulted in a dose-dependent increase of cAM P release by SaOS-2 cells into the effluent medium. Cyclic AMP accumul ation was rapidly desensitized by approx. 80% after 30 min of continuo us exposure to PTH(1-34) (10(-7) M), while cells remained responsive t o forskolin. The recovery of PTH responsiveness required at least 2 h of hormone-free perifusion. Desensitization in the experimental settin g was dose-dependent (EC(50) = 1.10(-10) M PTH(1-34)). Neither 8Br-cAM P (2.10(-4) M) nor PMA (1.10(-7) M) had an effect on the PTH(1-34)-ind uced desensitization of the adenylate cyclase. Radioreceptor assays sh owed that [I-125]-[Tyr(36)]hPTHrP(1-36)amide binding to SaOS-2 cells w as 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 n o effect on radioligand binding. PMA (1.10(-7) M) appeared to slightly increase [I-125]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 th e recovery from receptor internalization. We conclude that a perifusio n 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 ob served that does not appear to involve protein kinase A or C.