Comparative Study On 5-HT_1B Receptor And 5-HT Transporter Mechanism Of 5-HT-induced PASMC_s Proliferation

IntroductionPulmonary hypertension (PHT) is characterized by persistent vasoconstriction , structural remodeling of pulmonary vessels, inflammation and thrombosis. Hyperplasia of pulmonary artery smooth muscle cells (PASMCs) is the main component of pulmonary vessel remodeling that results in PHT. Many studies have shown that 5 - hydroxytryptamine (5 - HT) may be a key determinant of pulmonary remodeling, but the exact mechanism of proliferation of PASMCS has not been fully elicited.It was reported that there was an increased expression of 5 - HT1B receptor mRNA in monocrotaline (MCT) induced PHT rats. In wild - type mice, hypoxia increased right ventricular hypertrophy, pulmonary vascular remodeling, which was absent in 5 - HT1B receptor knockoutmice. 5 - HT transporter (5 -HTT) mediates internalization of indoleamine. Exposure of PASMCs to hypoxia results in a rapid increase 5 - HTT expression and activity, and this effect associated with potentiation of the mitogenic action of 5 - HT. It suggested that 5 -HT1B receptor and 5 - HTT play key role in pulmonary vessel remodeling.The present studies showed that 5 - HT induced the mitogen - activated protein kinases (MAPK) extracelluar signal -regulated kinase1 (ERK1) and ERK2 in rabbit renal artery smooth muscle cells. But in rat PASMCS, whether the intracelluar signal pathway of 5 - HT is dependent on ERKl/2 pathway is unconcerned. So, the aim of the present study was to investigate the receptor and the transporter mechanism of PASMCS proliferation and mitogenesis induced by 5 - HT and the intracelluar signal pathway of 5 - HT.Material and methodLiposomal transfection was used to introduce ODNs to ERK1/2 into cultured rat PASMCs and the transfection efficiency was measured by observing the uptake of the fluorecein isothiocynate ( FITC) - labeled antisense ODN in PASMCs. The effects of SB224289 - HT1B receptor selective inhibitor) , fluoxetine (5 - HIT selective inhibitor) and ODNs on the proliferation of PASMCs induced by 5 - HT and sumatriptan were evaluated by cells number counting and cell cycle analysis, and measured by Microculture tetrazolium (MTT) assay and Flow cytometry (FCM) respectively.Results1. Uptake of ODNs by PASMCs24 h after liposomal transfection, FITC - labeled antisense ODN were observed in both cytoplasm and the nuclei of PASMCs. There were more than 90% of the cells exhibited fluorescence.2. Microculture tetrazolium ( MTT) assay① The effect of SB224289, fluoxetine, SB224289 adding fluoxetine, ODNs on the proliferation of PASMCs induced by 5 - HT (1 μmol/L). The proliferation rate (PR) in response to 5 - HT increased from 130. 33% ±5. 24% to 195. 67% ±19.46%. 88224289(1 μmol/L, 100 nmol/L) , fluoxetine (1μmol/L, 100 nmol/L) , 86224289(1 μmol/L, 100 nmol/L) adding fluoxetine (1 μmol/L, 100 nmol/L) concentration - dependently inhibited the proliferation of PASMCs induced by 5 - HT( 1 μmol/L) , the PR decreased froml28.67% ± 13. 25% , 123.33% ±9.94%, 105.67% ±5.55% to 139.33% ±8.95%, 140.00% ± 3.21%, 124% ±7.64% respectively. Antisense ODN inhibited the proliferation of PASMCs significantly. And this ihibitory effect is similar to SB224289 adding fluoxetine. Sense ODN and random ODN showed no effect. ② Antisense ODN inhibited the proliferation of PASMCs induced by sumatriptan (1 μmol/L) in vitro from 164.67% ±6.67% to 76.67% ± 10.17%. Sense ODN and randomODN showed no effect.3. Flow cytometry (FCM)Flow cytometric analysis of cycle distribution showed that cells treated with SB224289, fluoxetine, SB224289 adding fluoxetine had litter values of SPF and PI than 5 - HT. The SPF decreased from 13% ±0.58% to 8.67% ±0.33% , 10. 33% ±0.33% , 6.33% ±0.33%. The PI decreased from 28.67% ±0.33% , to 25.67% ±0.33% , 26.33% ±0.88% , 23.67% ±0.33%. The increase of sumatriptan induced SPF and PI were significantly inhibited by ASODN with SPF from 11.67% ±0.33% to 3.33% ±0.33%, and PI from 27.33% ±0.33% to 22% ±0.58%.DiscussionThe present studies demonstrated that there is 5 - HT1B receptor mechanism and 5 - HTT mechanism on the proliferation of rat PASMCS induced by 5 - HT. We proposed the intracellular signal pathway of 5 - HT is ERK1/2 pathway dependent. Furthermore, 5 - HT activated ERKl/2 through 5 - HT1B receptor and 5-HTT.The data of present studies confirmed SB224289 ( a selective inhibitor of 5 -HT1B receptor) and fluoxetine (a selective inhibitor of 5 - HTT) inhibited the proliferation of PASMCs induced by 5 - HT. Keegan et al found that GR127935 ( a selective inhibitor of 5 - HT1B receptor) attenuated right ventricular pressure, right ventricular remodeling, vascular remodeling in CHPHT rats. The results of our studied proved sumatriptan induced the proliferation of PASMCs significantly. Some scholars assessed the role of 5 - HTT in mediating the mitogenic and comitogenic effects of 5 - HT in PASMCS. Precious studies demonstrated that in mice lacking the 5 - HTT gene and exposed to hypoxia, the remodeling of pulmonary vessels were decreased as wild - type controls. The evidence supported the key role for 5 - HT1B receptor and 5 - HTT in PASMCS proliferation and PHT.Activation of ERKl/2 pathway mediates cellular mitogenesis. In the present studies, the antisense ODN to ERKl/2 delivered by lipofectin proved an ef-...