Effects Of 5-Hydroxytryptamine On Coronary Artery Smooth Muscle Cell Proliferation, Migration And Sinoatrial Node Autorhythmicity

Part oneStudies for effects of 5-hydroxytryptamine on human coronary artery smooth muscle cell proliferation and migrationObjective To observe the effects of 5-hydroxytryptamine (5-HT) on human coronary artery smooth muscle cells (hCASMCs) viability and migration.Methods The hCASMCs were incubated with different concentrations of 5-HT (10-8M、 10-7M、10-6M、10-5M、10-4M、10-3M). The MTT assay was used to detect the effects of 5-HT on hCASMC proliferation, and transwell assay was used to dectect the effects of 5-HT on hCASMCs migration. Two 5-HT transporter (5-HTT) blockers, fluoxetine (10"5 mol/1) and citalopram (10-6 mol/1), and two 5-HT receptor (5-HTR) antagonists GR127935 (10-6 mol/1) and ketanserin (10-7 mol/1), were used to detect role of 5-HTT and 5-HTR on 5-HT induced hCASMCs proliferation and migration. High performance liquid chromatography was used to measure intracellular 5-HT. The hCASMCs were transfected with recombinant adenovirus containing the 5-HTT gene to confirm the role of 5-HTT on 5-HT induced hCASMCs proliferation and migration.Results (1) Most of the cells were typical fiber-like, spindle-shaped, and cell protrusions extending longer in contact with each other. The phenotype was characterized by smooth muscle a-actin staining. (2) MTT value of light absorption was markedly higher in 10-7 mol/1 group and top at 10-5 mol/1 (P<0.01), cell viability decreased when concentration is higher than 10-5 mol/1. (3) 5-HTT blockers inhibited 5-HT induced hCASMC proliferation and migration as well as 5-HT uptake (P<0.01). (4) 5-HTT overexpression increased 5-HT induced hCASMCs migration and proliferation (P<0.05).Conclusion 5-HT increased hCASMCs proliferation and migration, in which 5-HTT played an important role.Part two Expression and regulation of 5-hydroxytryptamine transporter during vascular injuryObjective Tp detect the expression of 5-hydroxytryptamine transporter (5-HTT) during vascular injury and its regulation by inflammatory factor.Methods Balloon denudation of the left common carotid artery of male Sprague-Dawley rats was performed. The arteries were collected at day 3,7,14 and 21 after balloon injury. Expressions of 5-HTT and interleukin-1 beta (IL-1β) were examined by western blotting. The hCASMCs were also treated by IL-1β (10 ng/ml) with or without IL-1 receptor antagonist (IL-1ra,10 ng/ml) to detect effect of IL-1β on 5-HTT expression and 5-HT induced current.Results (1) Neointimal formatted 7 days after sugery. After that, vascular smooth muscle cells and neointimal continued to grow. (2) The expression of IL-1β increased after balloon injury and peaked at 7 days (p<0.05), the expression of 5-HTT also increased after balloon injury and peaked at 14 days (p<0.05). (3) IL-1β pretreatment on hCASMC increased 5-HTT expression as well as 5-HT induced current on HEK293-5-HTT cells.Conclusion 5-HTT and IL-1β expression increased during restenosis; IL-1β functionally increased 5-HTT expression.Part three Serotonylation of cardiac myocytes and its effects on sinoatrial node autorhythmicityObjective To explore the target protein which can be serotonylized by 5-HT and to predict the effects on sinoatrial nodd autorhythmicity.Methods Rat cardic myocytes were isolated and randomly divided into four groups. Group A:myocytes were not pretreated by 5-HT and were treated by DSC and EGTA; group B: myocytes were pretreated by 1mM 5-HT and 6.7mM CaCl2; group C:myocytes were pretreated by DSC before coincubated with 5-HT; and group D:myocytes were pretreated by 5mM EGTA before co-incubated with 5-HT. Anti 5-HT antibody and Protein A Agarose were used to pull down the serotonylized protein for protein mass spectrometry analysis. The sino atrial node computer mathematics cell model was used to simulate the effects of serotonylized protein on sinus nodal autonomy.Results HPLC-mass spectral peptide sequencing revealed that a major protein serotonylated by 5-HT was sarco-endoplasmic reticulum calcium Atpase 2a (SERCA2a). The simulation results showed that decrease of SERCA2a forward calcium affinity, increase of SERCA2a reverse calcium affinity and mild sarco-endoplasmic reticulum (SR) calcium leak lead to fast pacing rate; while increase of SERCA2a forward calcium affinity, decrease of SERCA2a reverse calcium affinity and severe SR calcium leak lead to slow pacing rate.Conclusion SERCA2a was modified through serotonylation, and changes of SERCA calcium affinity lead to either tachycardia or bradycardia.