TY - JOUR
T1 - Neurotransmitter action in osteoblasts
T2 - Expression of a functional system for serotonin receptor activation and reuptake
AU - Bliziotes, M. M.
AU - Eshleman, A. J.
AU - Zhang, X. W.
AU - Wiren, K. M.
N1 - Funding Information:
This work was supported in part by the Medical Research Service of the Department of Veterans Affairs, and NIH Grant DK54415 to M.B. The authors gratefully acknowledge the excellent technical support and encouragement provided by Anne Chapman Evans and Les Alberque throughout the course of these studies. We also thank Dr. Laurie Vessely for assistance and critical reading of the manuscript.
PY - 2001
Y1 - 2001
N2 - Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2B receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT1A, 5-HT2A, and 5-HT2B receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a Km value of 1.13 μmol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.
AB - Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2B receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT1A, 5-HT2A, and 5-HT2B receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a Km value of 1.13 μmol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.
KW - Bone
KW - Neurotransmitter
KW - Osteoblast
KW - Receptor
KW - Serotonin
KW - Transporter
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U2 - 10.1016/S8756-3282(01)00593-2
DO - 10.1016/S8756-3282(01)00593-2
M3 - Article
C2 - 11704501
AN - SCOPUS:0034747779
SN - 8756-3282
VL - 29
SP - 477
EP - 486
JO - Bone
JF - Bone
IS - 5
ER -