Neuroendocrine regulation of cyclic AMP formation in osteoblastic cell lines (UMR-106–01, ROS 17/2.8, MC3T3-E1, and Saos-2) and primary bone cells

Abstract

The effect of four different neuropeptides and norepinephrine (NE) on cyclic AMP formation in four different osteoblastic cell lines and in isolated neonatal mouse calvarial bone cells has been examined. In the rat osteosarcoma cell line UMR-106–01, vasoactive intestinal polypeptide (VIP, 0.001–1 μM), calcitonin gene-related peptide (CGRP, 0.3–30 nM), and NE (0.1–300 μM), but not neuropeptide Y (NPY, 0.001–1 μM) or substance P (SP, 0.1–10 μM), caused a dose-dependent stimulation of cyclic AMP formation. The stimulatory effects were synergistically potentiated by forskolin (0.1–3 μM). The effects of NE and VIP were time dependent, with an optimal effect seen at 5 minutes. The amount of cyclic AMP accumulated in cells stimulated with NE and VIP was in the same range. The amplitude of the cyclic AMP response induced by CGRP was smaller than that caused by VIP and NE. In the human osteosarcoma cell line Saos-2, NE (0.1 μM) and VIP (0.3 μM) stimulated cyclic AMP formation, and the effect was synergistically potentiated by forskolin. In the absence of forskolin, no effect of CGRP (30 nM) could be seen in the Saos-2 cells, but in the presence of forskolin (3 μM) a stimulatory effect was observed. SP and NPY did not change basal cyclic AMP levels in Saos-2 cells. In the osteoblastic osteosarcoma cell line of rat, ROS 17/2.8, NE (0.1 μM) caused a significant stimulatory action on cyclic AMP formation that was synergistically potentiated by forskolin (3 μM), VIP, CGRP, and SP did not affect the cellular content of cyclic AMP in ROS 17/2.8. In the mouse calvarial cell line MC3T3-E1 and in enzymatically isolated mouse calvarial bone cells, NE (0.1 μM), VIP (0.3 μM), and CGRP (30 nM), but not SP (10 μM) or NPY (1 μM), stimulated cyclic AMP formation. In UMR-106–01 cells and in isolated bone cells, but not in Saos-2 and ROS 17/2.8 cells, NPY (1 μM) inhibited the stimulatory effect of NE (0.3 μM) on cyclic AMP formation. These data show that osteoblastic cell lines are equipped with receptors for neuropeptides and NE. Since VIP, CGRP, NPY, and NE have been shown to occur in nerves of the skeleton, the observations in the present paper suggest the possibility that neurohormones may have a role in the regulation of osteoblastic activity.