Abstract
Aim: Human coronary artery smooth muscle cell (HCASMC) plays an important role in the pathogenesis of coronary atherosclerosis and coronary artery diseases (CAD). Serotonin is a mediator known to produce vascular smooth muscle cell (VSMC) mitogenesis and contribute to coronary atherosclerosis. We hypothesize that the human coronary artery smooth muscle cell possesses certain vital elements of the serotonergic system, viz. tryptophan hydroxylase and serotonin transporter, which play a critical role in the synthesis and VSMC-mitogenic action of serotonin, respectively. Our aim was to demonstrate the presence of functional tryptophan hydroxylase 1 and serotonin transporter in HCASMC.|Methods: HCASMCs from passages 3 -6 were used for the experiments. The messenger ribonucleic acid (mRNA) transcripts and protein expression of tryptophan hydroxylase1 (TPH1) and serotonin transporter (SERT) were examined by real-time PCR (Real time polymerase chain reaction) and western blot analysis, respectively. The specificity and accuracy of the primers was verified using DNA (Deoxyribonucleic acid) gel electrophoresis and sequencing of real-time PCR products. The functionality of SERT was examined using a fluorescence dye based serotonin transporter assay and the enzymatic activity of TPH was demonstrated using Ultra Performance Liquid Chromatography (UPLC). |Results: mRNA transcripts and protein of both SERT and TPH are expressed in HCASMCs. The real-time PCR showed a single melt curve peak for both transcripts, and the amplicons when sequenced aligned with the respective genes. The Western blots revealed distinct bands for SERT and TPH at 70 KDa and 50 KDa, respectively. Serotonin transporter activity was present in HCASMCs. UPLC of the HCASMC lysate, to which the substrates and catalysts for the TPH enzyme had been provided, revealed the presence of 5-hydroxy tryptophan. Conclusion: Both tryptophan hydroxylase and serotonin transporter are expressed in HCASMCs. The expressed proteins are functional. These findings are novel and an initial step in examining the clinical relevance of the serotonergic system in HCASMC and its role in the pathogenesis of coronary atherosclerosis and CAD