Neurotransmitter action in osteoblasts: Expression of a functional system for serotonin receptor activation and reuptake

M. M. Bliziotes, A. J. Eshleman, X. W. Zhang, Kristine Wiren

Research output: Contribution to journalArticle

171 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)477-486
Number of pages10
JournalBone
Volume29
Issue number5
DOIs
StatePublished - 2001

Fingerprint

Serotonin Receptors
Osteoblasts
Neurotransmitter Agents
Serotonin
Receptor, Serotonin, 5-HT2B
Imipramine
Fluoxetine
Binding Sites
Parathyroid Hormone
Norepinephrine Plasma Membrane Transport Proteins
Bone and Bones
Dopamine Plasma Membrane Transport Proteins
Desipramine
Staurosporine
Dopamine Antagonists
Transcription Factor AP-1
Morphogenesis
Cocaine
Inhibitory Concentration 50
Reverse Transcription

Keywords

  • Bone
  • Neurotransmitter
  • Osteoblast
  • Receptor
  • Serotonin
  • Transporter

ASJC Scopus subject areas

  • Physiology
  • Hematology

Cite this

Neurotransmitter action in osteoblasts : Expression of a functional system for serotonin receptor activation and reuptake. / Bliziotes, M. M.; Eshleman, A. J.; Zhang, X. W.; Wiren, Kristine.

In: Bone, Vol. 29, No. 5, 2001, p. 477-486.

Research output: Contribution to journalArticle

Bliziotes, M. M. ; Eshleman, A. J. ; Zhang, X. W. ; Wiren, Kristine. / Neurotransmitter action in osteoblasts : Expression of a functional system for serotonin receptor activation and reuptake. In: Bone. 2001 ; Vol. 29, No. 5. pp. 477-486.
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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.

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