Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells.
Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 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 MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 microM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.
Bliziotes
M
M
Portland VA Medical Center, Portland, OR 97239, USA. bliziote@ohsu.edu
Eshleman
A
A
Burt-Pichat
B
B
Zhang
X-W
XW
Hashimoto
J
J
Wiren
K
K
Chenu
C
C
D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D015870:Gene Expression; D007150:Immunohistochemistry; D007700:Kinetics; D051379:Mice; D010006:Osteoblasts / Q000378:metabolism; D010011:Osteocytes / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D044282:Receptor, Serotonin, 5-HT1A / Q000235:genetics* / Q000378:metabolism*; D044402:Receptor, Serotonin, 5-HT2A / Q000235:genetics* / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050486:Serotonin Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D013977:Tibia / Q000378:metabolism
D012333:RNA, Messenger; D044402:Receptor, Serotonin, 5-HT2A; D050486:Serotonin Plasma Membrane Transport Proteins; C493231:Slc6a4 protein, mouse; C493232:Slc6a4 protein, rat; D044282:Receptor, Serotonin, 5-HT1A